Reverse Transcriptase Mutants with Increased Activity and Thermostability

ABSTRACT

The disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure as provides suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 17/380,982, filed on Jul. 20, 2021, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/054,228, filed on Jul. 20, 2020. Each of the above-identified applications are hereby incorporated by reference herein in their entireties for all purposes.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically as a text file in ASCII format and is hereby incorporated by reference in its entirety. The name of the ASCII text file is “20-1076-US-CIP_Sequence-Listing_ST25_FINAL.txt.”

FIELD OF THE DISCLOSURE

The disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure also relates to suitable amino acid positions in MMLV RTase for mutagenesis and methods for using MMLV RTase mutants to synthesize cDNA from RNA templates.

BACKGROUND

Reverse transcriptase (RTase) enzymes have revolutionized molecular biology. RTase is a critical component of the reverse transcription polymerase chain reaction (RT-PCR) allowing the production of complementary DNA (cDNA) from RNA. The cDNA produced in reverse transcription reactions can be used in a wide range of downstream applications, including quantitative PCR, gene expression analysis, isolated RNA sequencing, gene cloning, and cDNA library creation.

RTases, first derived from retroviruses, facilitate the reverse transcription of RNA into cDNA by utilizing RNA-dependent polymerase and RNase H, a non-sequence-specific endonuclease enzyme that catalyzes cleavage of RNA in an RNA/DNA duplex. This results in virus replication and integration of the viral sequence into host DNA thereby allowing for the proliferation of the virus along with host DNA. Within the laboratory setting, RTases from Moloney murine leukemia virus (MMLV), avian myeloblastosis virus (AMV), and human immunodeficiency virus type 1 (HIV-1) are the most commonly used RTase for cDNA synthesis.

RTases for research applications are often mutated multi-generational MMLV and AMV RTases that have been optimized for laboratory procedures. Mutations in the RTases alter properties of the enzymes, including thermostability, RTase activity, 5′ mRNA coverage, and RNase H activity.

AMV RTases are thermostable and less sensitive to thermal degradation than MMLV RTase and are preferred for RNA having a strong secondary structure. In addition, AMV RTases are often suitable for use with RNA molecules that are five kilobases or longer because of the heat stability of AMV RTases. However, the high temperatures required to resolve strong secondary structures or long RNA strands can negatively impact RNA integrity and fidelity of transcription. AMV also possess an intrinsic RNase activity that degrades RNA in an RNA/DNA hybrid, which can result in reduced total cDNA and reduced full-length cDNA yield.

MMLV RTase is characterized by low RNase H activity and a higher fidelity as compared to AMV RTase. The reduced RNase H activity allows MMLV RTases to be used for the reverse transcription of long RNAs (>5 kb). However, the RNase H activity of MMLV RTase limits the efficiency of synthesizing long cDNA in vitro. Mutations in MMLV RTase have been introduced to reduce RNase H activity. In addition, because the optimal temperature for MMLV RTase activity is ˜37° C., the enzyme lacks the ability to effectively reverse transcribe RNAs with strong secondary structures. The use of MMLV RTase at elevated temperatures can compromise cDNA length and yield as a result of lower enzyme activity. MMLV RTase mutants that substitute Mn²⁺ for Mg²⁺ in the reaction mixture attempt to overcome these limitations, but are characterized by inefficiency and error.

Thus, despite the unique properties of AMV and MMLV RTases, there exists a need for an RTase that combines the beneficial attributes of AMV and MMLV RTases. Consistent with this, the present application discloses MMLV RTase mutants, isolated through rational mutagenesis of MMLV RTase, that exhibit increased RTase activity and thermostability as compared to RTases, including RNase H minus constructs, that are currently available in the art.

SUMMARY

The disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure also provides suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.

One aspect of the disclosure provides an isolated Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutant comprising the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) threonine to lysine at position 306 (T306K); (i) a valine to asparagine at position 433 (V433N); (j) a valine to arginine at position 433 (V433R); (k) an isoleucine to glutamic acid at position 593 (1593E); or (1) an isoleucine to tryptophan at position 593 (I593W).

Another aspect of the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an MMLV RTase mutant of the disclosure.

Other aspects of the disclosure provide a composition or a kit comprising an MMLV RTase mutant of the disclosure.

Other aspects of the disclosure provide methods for synthesizing complementary deoxyribonucleic acid (cDNA) or methods for performing reverse transcription-polymerase chain reaction (RT-PCR) using an MMLV RTase mutant of the disclosure.

Specific embodiments of the disclosure will become evident from the following more detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are schematics showing reverse transcriptase mutagenesis selection by rational design. Amino acid positions for mutagenesis were chosen at the substrate binding site (FIGS. 1A and 1B) or near the substrate binding site (FIG. 1C).

FIG. 2 shows Western blot analysis of test induction results in in BL21(DE3) cells for MMLV RT in TB medium. Lane 1—Precision Plus Protein Unstained Standards (Bio Rad, Cat #161-0363), Lane 2—Time=0 hour, Lane 3—Time=3 hours after induction at 37° C., Lane 4—Time=0 hour, Lane 5—Time=21 hours after induction at 18° C.

DETAILED DESCRIPTION

The disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure also relates to suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.

The MMLV RTase mutants of the disclosure, which have been identified and isolated, at least in part, through rational mutagenesis of a base construct of MMLV RTase, were found to have increased RTase activity and thermostability as compared to wild-type MMLV RTase and certain MMLV RTase mutants, including RNase H minus RTases, that are currently available in the art.

Reference will now be made in detail to exemplary embodiments of the claimed invention. While the claimed invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the claimed invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents, as may be included within the spirit and scope of the claimed invention, as defined by the appended claims.

Those of ordinary skill in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the claimed invention. In addition, although certain methods and materials are described herein, other methods and materials that are similar or equivalent to those described herein can also be used to practice the claimed invention.

In addition, any of the compositions or methods provided, disclosed, or described herein can be combined with one or more of any of the other compositions and methods provided, disclosed, or described herein.

1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the claimed invention belongs. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the claimed invention. All technical and scientific terms used herein have the same meaning.

The following references provide those of skill in the art with a general understanding of many of the terms used herein (unless defined otherwise herein): Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd ed. (Wiley, 2006); Walker, The Cambridge Dictionary of Science and Technology (Cambridge University Press, 1990); Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed. (Springer Verlag, 1991); and Hale et al., Harper Collins Dictionary of Biology (HarperCollins Publishers, 1991). Generally, the procedures or methods described herein and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as, for example, Green et al., Molecular Cloning: A Laboratory Manual, 4th ed. (Cold Spring Harbor Laboratory Press, 2012), and Ausubel, Current Protocols in Molecular Biology (John Wiley & Sons Inc., 2004).

The following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings known or understood by those having ordinary skill in the art are also possible, and within the scope of the claimed invention. All publications, patent applications, patents, and other references mentioned or discussed herein are expressly incorporated by reference in their entireties. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

As used herein, the singular forms “a,” “and,” and “the” include plural references, unless the context clearly dictates otherwise.

As used herein, the term “or” means, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

As used herein, the term “including” means, and is used interchangeably with, the phrase “including but not limited to.”

As used herein, the term “such as” means, and is used interchangeably with, the phrase “such as, for example” or “such as but not limited.”

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example, within two standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “nucleic acid molecule” and “polynucleotide” refer to a polymer or large biomolecule comprised of nucleotides. The term “nucleic acid” includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogs thereof. Non-limiting examples of nucleic acid molecules include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA, rRNA, cRNA, tRNA), and chimeras thereof. A nucleic acid molecule can be obtained by cloning techniques or synthesized, using techniques that are known to those of skill in the art. DNA can be double-stranded or single-stranded (coding strand or non-coding strand, i.e., antisense). A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as “peptide nucleic acids” (PNA)), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, for example, 2′ methoxy substitutions (containing a 2′-O-methylribofuranosyl moiety) and/or 2′ halide substitutions. Nitrogenous bases may be conventional bases (adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U)), known analogs thereof (e.g., inosine), known derivatives of purine or pyrimidine bases, or “abasic” residues in which the backbone includes no nitrogenous base for one or more residues. A nucleic acid may comprise only conventional sugars, bases, and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs). An “isolated nucleic acid molecule,” as is generally understood by those of skill in the art and as used herein, refers to a polymer of nucleotides, and includes but is not limited to DNA and RNA.

As used herein, the term “probe” refers to a nucleic acid oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's “target” generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or “base pairing.” Sequences that are “sufficiently complementary” allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled. A probe can be produced by molecular cloning of a specific DNA sequence or it can be synthesized. Probes for use in the methods disclosed herein can be readily designed and used by those of skill in the art.

As used herein, the term “primer” refers to a nucleic acid oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid or its complement, and which is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Primers may be provided in double-stranded or single-stranded form. Primers for use in the methods disclosed herein can be readily designed and used by those of skill in the art.

Probes or primers for use in the methods disclosed herein may be of any suitable length, depending on the particular assay format and the particular needs and targeted sequences employed. For example, the probes or primers for use in the methods disclosed herein are at least 10 nucleotides in length, or at least 15, 20, 25, 30, or more than 30 nucleotides in length, and they may be adapted to be especially suited for a chosen nucleic acid amplification system and/or hybridization system used. Longer probes and primers are also within the scope of the disclosure.

A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analog of such RNA or cDNA) that is complementary to or having a high percentage of identity (e.g., at least 80% identity) with all or a portion of a mature mRNA made by transcription of a marker of the disclosure and normal post-transcriptional processing (e.g., splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

As used herein, the terms “reverse transcriptase,” “RTase,” or “RT” refer to an enzyme that is used to generate complementary (cDNA) from an RNA template in a process known as “reverse transcription.” The term reverse transcriptase, as used herein, also refers to any enzyme that exhibits reverse transcription activity. Reverse transcriptases can be derived from a variety of sources including but not limited to viruses including retroviruses and DNA polymerases exhibiting transcriptase activity. Such retroviruses include but are not limited to Moloney murine leukemia virus (MMLV), avian myeloblastosis virus (AMV), and human immunodeficiency virus (HIV).

Reverse transcriptase activity can be measured by incubating an RTase in a buffer containing an RNA template and deoxynucleotides. One of skill in the art will recognize that a wide range of conditions can be used to perform reverse transcription reactions and multiple methods exist for measuring the quantity of cDNA produced during reverse transcription.

Reverse transcriptases of the disclosure include reverse transcriptases having one or a combination of the properties described herein. Such properties include but are not limited to increased activity, enhanced DNA synthesis, enhanced stability or enhanced thermostability, reduced or eliminated RNase H activity, reduced terminal deoxynucleotidyl transferase activity, increased accuracy or increased fidelity, increased specificity, or altered half-life, for example when compared to a base construct. As used herein, the term “base construct” refers to the initial RTase from which the RTase mutants of the disclosure are prepared (e.g. for example a wild-type RTase or a modified wild-type RTase).

As used herein, the terms “accuracy” and “fidelity” are used interchangeably and refer to ability of an RTase to accurately replicate a desired template; i.e., the ability of the RTase to accurately perform cDNA synthesis in a reverse transcription reaction. The “fidelity” or “accuracy” of a reverse transcriptase can be assessed by determining the frequency of incorrect nucleotide incorporation into the synthesized cDNA molecule, which may be referred to as the enzyme's error rate. As used herein, the term “increased fidelity” refers to RTase mutants of the disclosure that exhibit an error rate lower than that of the base construct. For example, the RTase mutants as disclosed herein can exhibit an error rate that is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 100%, or 200% lower than, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, or more than 10-fold lower than the error rate of the RTase base construct . . . .

As used herein, the term “specificity” refers to a decrease in mis-priming by an RTase during cDNA synthesis. An RTase mutant's specificity can be assessed by performing a reverse transcription reaction at a particular temperature, including higher temperatures, and comparing the amount of mis-priming in that reaction with the amount of mis-priming in a reaction performed with the wild-type RTase (or the RTase base construct) under identical conditions.

As used herein with respect to the RTase molecules of the disclosure, the terms “stable” and “thermostable” are used interchangeably and refer to an enzyme that is resistant to heat inactivation and remains active at temperatures in excess of 37° C. (e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 70° C., or higher temperatures). For example, in one embodiment the disclosure provides an RTase mutant having activity with a longer half-life than that of the base construct RTase at an elevated temperature. Thus, RTase mutants with “enhanced thermostability” can refer to RTase mutants of the disclosure that exhibit an increase in thermostability at temperatures of about 50° C. up to about 90° C. as compared to the base construct RTase. In some embodiments, the thermostability of the RTase mutant is at least 1.5 fold or greater as compared to the thermostability of the base construct RTase. Comparisons of cDNA produced by a base construct and RTase mutant are compared using identical reaction conditions for the base construct and RTase mutant reactions. Reaction conditions can include but are not limited to salt concentration, buffer concentration, pH, divalent metal ion concentration, temperature, nucleoside triphosphate concentration, template concentration, RTase concentration, primer concentration, time, and in one-step PCR, the quantitative PCR primer and probe concentrations.

As used herein, the term “enhanced DNA synthesis” refers to an RTase enzyme that produces more DNA (e.g. cDNA) than the base RTase construct. In some embodiments, DNA synthesis can be measured by quantitative PCR at standard reaction conditions, as compared to the base construct RTase. Consistent with assessments of thermostability, quantitative comparisons are made under similar or the same reaction conditions and the amount of cDNA synthesized using the base construct RTase is compared to the amount of cDNA produced using the RTase mutant (see Tables 4-7). In some embodiments, the RTase mutant of the disclosure with enhanced DNA synthesis may produce about 5% to about 200% more cDNA than the base construct RTase. In some embodiments, the RTase mutant of the disclosure with enhanced DNA synthesis has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or 200% more than, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, or more than 10-fold more DNA synthesis than the RTase base construct DNA synthesis.

Reverse transcriptase activity, as described herein, was evaluated in a one-step or two-step procedure. The one-step procedure combines reverse transcription and quantitative PCR in a single reaction. The method is performed by including Gene Expression Master Mix, RTase, RNA, a fluorescent probe, and primers and probes as described in Example 3. The two-step procedure comprises reverse transcription followed by quantitative PCR. In the reverse transcription step, RTase is added to a mixture containing RNA, gene specific primers, first strand synthesis buffer, and RNase. The resultant cDNA is then quantified in a second step wherein the cDNA is combined with Gene Expression Master Mix, primers and probes, and a fluorescent marker. The cDNA produced in either the one-step and two-step procedures is quantified, and the mean and standard deviation reported as shown herein in Tables 4-7.

As used herein, “RNase H activity” refers to cleavage of RNA in DNA-RNA duplexes via a hydrolytic mechanism to produce 5′ phosphate terminated oligonucleotides. RNase H activity does not include degradation of single-stranded nucleic acids, duplex DNA, or double-stranded RNA. As used herein, the phrase “substantially lacks RNase H activity” means having less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wild type enzyme. As used herein, the phrase “lacks RNase H activity” means having undetectable RNase H activity or having less than about 1%, 0.5%, or 0.1% of the RNase H activity of a wild type enzyme.

As used herein, the term “mutation” refers to a change introduced into the nucleic acid sequence encoding a protein that changes the amino acid sequence of the protein, including but not limited to substitutions, insertions, deletions, point mutations, transpositions, inversions, frame shifts, nonsense mutations, truncations, or other forms of aberrations. A mutation may produce no discernible changes or result in a new property, function, or trait of the mutated protein. An RTase mutant of the disclosure may have one or more mutations in the nucleic acid sequence encoding the RTase mutant resulting in one or more mutations in the amino acid sequence of the RTase mutant. A mutation can result in one or more amino acids being substituted for an alternate amino acid residue, including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and/or Val. The resulting amino acid mutations may impart altered functional and biological properties to the RTase mutant including but not limited to increased activity, enhanced DNA synthesis, enhanced stability or enhanced thermostability, reduced or eliminated RNase H activity, reduced terminal deoxynucleotidyl transferase activity, increased accuracy or increased fidelity, increased specificity, or altered half-life.

As used herein, the terms “detecting,” “detection,” “determining,” and the like refer to assays performed for identification of the quantity of cDNA synthesis as a marker of RTase activity. The amount of marker expression or activity detected in the sample can be the same as, decreased, or increased as compared to the amount of marker expression or activity detected using the RTase base construct. One of skill in the art will understand that amount of cDNA can be quantified using multiple techniques.

The term “increased,” as used herein with regard to RTase activity, refers to the level of RTase activity of an RTase mutant as compared to the RTase base construct. An RTase mutant has “increased” RTase activity if the level of its RTase activity, as measured by the quantity of cDNA synthesized or as measured by other methods known in the art, is more than the RTase base construct activity. For example, the RTase activity of the RTase mutant is increased if the RTase activity is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, or more than 10-fold more than the RTase base construct activity.

The term “decreased,” as used herein with regard to RTase activity, refers to the level of RTase activity of an RTase mutant as compared to the RTase base construct. An RTase mutant has “decreased” RTase activity if the level of its RTase activity, as measured by the quantity of cDNA synthesized or as measured by other methods known in the art is less than the RTase base construct activity. For example, the RTase activity of the RTase mutant is decreased if the RTase activity is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% less than, or at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more than 10-fold less than the RTase base construct activity.

As used herein, the term “amplification” refers to any known in vitro procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. Known in vitro amplification methods include, for example, transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification, and strand-displacement amplification (SDA, including multiple strand-displacement amplification method (MSDA)). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Q-β-replicase. PCR amplification uses DNA polymerase, primers, and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA. PCR involves denaturation of a double-stranded DNA molecule, followed by annealing of DNA primers directed to the sequence of interest, and amplification/extension of the newly formed DNA strand. LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation. SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Other strand-displacement amplification methods known in the art (e.g., MSDA) do not require endonuclease nicking. Those of skill in the art will understand that the oligonucleotide primer sequences of the disclosure may be readily used in any in vitro amplification method based on primer extension by a polymerase. As commonly known in the art, oligonucleotides are designed to bind to a complementary sequence under selected conditions.

As used herein, “real time PCR” or “quantitative PCR” refers to a PCR method wherein the amount of product being formed can be monitored using florescent probes and quantified by tracking the fluorescent signal produced, above a threshold level. Real time PCR can be performed in a one-step reaction that includes the reverse transcription step in a simultaneous reaction (i.e., real time PCR or RT-PCR) or in a two-step reaction in which the reverse transcription step and PCR steps are performed consecutively.

As used herein, the term “complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a nucleotide of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides of the first portion are capable of base pairing with nucleotides in the second portion. In another embodiment, all nucleotides of the first portion are capable of base pairing with nucleotides in the second portion.

Polypeptide and polynucleotide sequences may be aligned, and percentages of identical amino acids or nucleotides in a specified region may be determined against another polypeptide or polynucleotide sequence, using computer algorithms that are publicly available. The percent identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the disclosure; and then multiplying by 100 to determine the percent identity.

As used herein, the terms “sample” and “biological sample” include a specimen or culture obtained from any source. Biological samples can be obtained from cerebrospinal fluid, lacrimal fluid, blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), urine, saliva, and the like. Biological samples also include tissue samples, such as biopsy tissues or pathological tissues that have previously been fixed (e.g., formaline snap frozen, cytological processing).

2. Reverse Transcriptases

The disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The MMLV RTase mutants of the disclosure are prepared by modifying the sequence of an MMLV RTase base construct (SEQ ID NO: 637). In one embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least one amino acid substitution that is: (a) an isoleucine to arginine, lysine, or methionine substitution at position 61 (I61R, I61K, or I61M); (b) a glutamine to arginine, lysine, or isoleucine substitution at position 68 (Q68R, Q68K, or Q68I); (c) a glutamine to arginine, histidine, or isoleucine substitution at position 79 (Q79R, Q79H, or Q79I); (d) a leucine to arginine, lysine, or asparagine substitution at position 99 (L99R, L99K, or L99N); (e) a glutamic acid to aspartic acid, methionine, or typtophan substitution at position 282 (E282D, E282M, or E282W); and/or (f) an arginine to alanine substitution at position 298 (R298A).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) an isoleucine to arginine substitution at position 61 and a glutamic acid to aspartic acid substitution at position 282 (I61R/E282D); (b) a leucine to arginine at substitution position 99 and a glutamic acid to aspartic acid substitution at position 282 (L99R/E282D); (c) a glutamine to arginine substitution at position 68 and a glutamic acid to aspartic acid substitution at position 282 (Q68R/E282D); (d) a glutamine to arginine substitution at position 79 and a glutamic acid to aspartic acid substitution at position 282 (Q79R/E282D); (e) a glutamic acid to aspartic acid substitution at position 282 and an arginine to alanine substitution at position 298 (E282D/R298A); (f) an isoleucine to arginine substitution at position 61 and a leucine to arginine substitution at position 99 (I61R/L99R); (g) an isoleucine to arginine substitution at position 61 and a glutamine to arginine substitution at position 68 (I61R/Q68R); (h) an isoleucine to arginine substitution at position 61 and a glutamine to arginine substitution at position 79 (I61R/Q79R); (i) an isoleucine to arginine substitution at position 61 and an arginine to alanine substitution at position 298 (I61R/R298A); (j) a glutamine to arginine substitution at position 68 and a leucine to arginine substitution at position 99 (Q68R/L99R); (k) a glutamine to arginine substitution at position 79 and a leucine to arginine substitution at position 99 (Q79R/L99R); (1) a leucine to arginine at substitution position 99 and an arginine to alanine substitution at position 298 (L99R/R298A); (m) a glutamine to arginine substitution at position 68 and a glutamine to arginine substitution at position 79 (Q68R/Q79R); (n) a glutamine to arginine substitution at position 68 and an arginine to alanine substitution at position 298 (Q68R/R298A); or (o) a glutamine to arginine substitution at position 79 and an arginine to alanine substitution at position 298 (Q79R/R298A).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least three amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/L99R/E282D); (b) a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q79R/L99R/E282D); (c) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 68, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/E282D); or (d) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 68, and a leucine to arginine substitution at position 99 (Q68R/Q79R/L99R).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least four amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/L99R/E282D); (b) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to lysine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/L99K/E282D); (c) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to asparagine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/L99N/E282D); (d) a glutamine to isoleucine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68I/Q79R/L99R/E282D); (e) a glutamine to lysine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68K/Q79R/L99R/E282D); (f) a glutamine to arginine substitution at position 68, a glutamine to histidine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79H/L99R/E282D); (g) a glutamine to arginine substitution at position 68, a glutamine to isoleucine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79I/L99R/E282D); (h) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to methionine substitution at position 282 (Q68R/Q79R/L99R/E282M); (i) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to tryptophan substitution at position 282 (Q68R/Q79R/L99R/E282W); or (j) a glutamine to isoleucine substitution at position 68, a glutamine to histidine substitution at position 79, a leucine to lysine substitution at position 99, and a glutamic acid to methionine substitution at position 282 (Q68I/Q79H/L99K/E282M).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least five amino acid substitutions that are: (a) an isoleucine to lysine substitution at position 61, a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (I61K/Q68R/Q79R/L99R/E282D); (b) an isoleucine to methionine substitution at position 61, a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (I61M/Q68R/Q79R/L99R/E282D); or (c) an isoleucine to methionine substitution at position 61, a glutamine to isoleucine substitution at position 68, a glutamine to histidine substitution at position 79, a leucine to lysine substitution at position 99, and a glutamic acid to methionine substitution at position 282 (I61M/Q68IR/Q79H/L99K/E282M).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least five or more amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a valine to arginine substitution at position 433, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E): (b) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 82, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a valine to arginine substitution at position 433, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E); (c) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 82, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a threonine to glutamic acid substitution at position 332, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E); (d) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 82, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a threonine to glutamic acid substitution at position 332, a valine to arginine substitution at position 433, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) threonine to lysine at position 306 (T306K); (i) a valine to asparagine at position 433 (V433N); (j) a valine to arginine at position 433 (V433R); (k) an isoleucine to glutamic acid at position 593 (I593E); or (1) an isoleucine to tryptophan at position 593 (I593W).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises the amino acid substitutions: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine substitution at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine substitution at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) a threonine to lysine substitution at position 306 (T306K); (i) a valine to asparagine substitution at position 433 (V433N); and (j) an isoleucine to tryptophan substitution at position 593 (I593W).

In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises the amino acid substitutions: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine substitution at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine substitution at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) a threonine to lysine substitution at position 306 (T306K); (i) a valine to arginine substitution at position 433 (V433R); and (j) an isoleucine to glutamic acid substitution at position 593 (I593E).

In one embodiment the RTase mutant amino acid sequence comprises a mutant selected from Tables 3, 8, 9, 12, 21, 22, or 38. In one aspect, the RTase mutant amino acid sequence comprises a mutant selected from the amino acid sequences of SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 679, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, or SEQ ID NO: 731.

In one embodiment the RTase mutant amino acid sequence comprises a C-terminal extension. In one aspect the C-terminal extension comprises a peptide sequence. In another embodiment an isolated polypeptide encodes a RTase mutant with a C-terminal extension

The claimed invention is based, at least in part, on the discovery that certain single and double amino acid mutations introduced into an MMLV RTase sequence, as disclosed herein, result in an MMLV RTase with increased or enhanced thermostability and/or RTase activity. Accordingly, methods for synthesizing the MMLV RTase mutants and methods for performing reverse transcription-polymerase chain reaction (RT-PCR) are also provided herein. Further provided are kits comprising the isolated MMLV RTase single, double, triple, or more mutations.

In certain embodiments, the mutated RTase is derived from the retrovirus Moloney murine leukemia virus (MMLV). In other embodiments, a mutated RTase of the disclosure could be derived from the RTase from a retrovirus other than MMLV, such as avian myeloblastosis virus (AMV) or human immunodeficiency virus type 1 (HIV-1), by introducing the same mutations into an RTase base construct obtained from the other retrovirus.

In certain embodiments, the RTase mutants of the disclosure are obtained by genetic engineering techniques that are well known in the art. For example, site-directed and random mutagenesis can be used to generate the RTase mutants of the disclosure.

In one embodiment of the disclosure, an RTase mutant of the disclosure is part of a composition.

3. Mutagenesis

The RTase mutants of the disclosure can be prepared by standard methods disclosed herein or known in the art. In one embodiment, the nucleic acid sequence of the RTase base construct (SEQ ID NO: 637) is modified to create a nucleic acid sequence encoding an RTase mutant. One of skill in the art will recognize that colonies with the appropriate strains can be used to grow and express an RTase mutant of interest, and following cell harvest and protein isolation, the RTase mutant can be used in cDNA synthesis techniques. Non-limiting examples of mutagenesis and cDNA synthesis are described herein in Examples 1-3.

As used herein, the term “mutagenesis” refers to the introduction of a genetic change in the nucleic acid sequence of a cell, wherein the alteration is then inherited by each cell. One of skill in the art will understand that mutations in a given nucleic acid sequence can be introduced using a variety of methods. One of skill in the art will further recognize that mutagenesis methods seek to mutate a target gene or target polynucleotide. The target gene may encode any one or more desired proteins. Mutagenesis methods commonly use a synthetic oligonucleotide that carries the desired sequence modification. The mutagenic oligonucleotide is incorporated into the DNA sequence using in vitro enzymatic DNA synthesis and is propagated in a mutant or wild-type bacterium.

Site directed mutagenesis, wherein targeted mutations are introduced into one or more desired positions of a template polynucleotide, may be achieved using primer extension mutagenesis. This technique requires the use of a specific primer that contains one or more desired mutations relative to the template polynucleotide. The mutagenesis primer can be a synthetic oligonucleotide or a PCR product. The mutated primer may include one or more substitutions, deletions, additions, or combinations thereof.

Mutated reverse transcriptases may also be generated using random mutagenesis, wherein mutations are introduced into the mutagenesis primer during synthesis. Randomly mutagenized oligonucleotides may also be used as mutagenesis primers.

In another embodiment, the mutated reverse transcriptases of the disclosure can be developed using error-prone rolling circle amplification (RCA). In this technique, the fidelity of a DNA polymerase is decreased by performing the RCA in the presence of MnCl₂ or by decreasing the amount of input DNA.

4. cDNA Synthesis

The disclosure also relates to the activity of MMLV RTases, as measured by the quantity of cDNA produced by the MMLV RTases disclosed herein. cDNA can be prepared using one-step or two-step procedures and can be obtained from a variety of template molecules. As used herein, the term “template molecule” refers to a biological molecule that carries the genetic code for use in making a new nucleic acid strand. For example, in DNA replication, the unwound double helix and each single-stranded DNA molecule is used as a template to synthesize a complementary strand. Reverse transcription generates cDNA from RNA. One of skill in the art will understand that cDNA molecules may be prepared from a variety of nucleic acid template molecules. In one embodiment, the nucleic acid template can be single-stranded or double-stranded DNA. In one embodiment, RNA can be used in cDNA synthesis. In certain embodiments, the MMLV RTase mutants of the disclosure exhibit increased or enhanced thermostability and/or RTase activity as compared to an RTase base construct. In other embodiments, the MMLV RTase mutants of the disclosure exhibit altered half-life, reduced or eliminated RNase H activity, reduced terminal deoxynucleotidyl transferase activity, increased accuracy or fidelity, or increased specificity.

The disclosure also provides methods for synthesizing cDNA using the MMLV RTase mutants of the disclosure that have single or double amino acid mutations. The MMLV RTase mutants of the disclosure may be used in methods that produce a first strand cDNA or a first and second strand cDNA. One of skill in the art will understand that first and second strand cDNA may form a double-stranded DNA molecule, which may include a full-length cDNA sequence and cDNA libraries.

The cDNA molecules that have been reverse transcribed by the MMLV RTase mutants of the disclosure may be isolated, or the reaction mixture containing the cDNA molecules may be directly used in downstream applications or for further analysis or manipulation. Amplification methods that may be used to practice the methods of the disclosure are described herein and are well known in the art. Reverse transcription reactions may be carried out using non-specific primers, such as an anchored oligo-dT primer, or random sequence primers, or using a target-specific primer complementary to the RNA for each genetic probe being monitored, or using thermostable DNA polymerases (such as AMV RTase or MMLV RTase).

Amplification methods utilize pairs of primers that selectively hybridize to nucleic acids corresponding to a specific nucleotide sequence of interest that are contacted with the isolated nucleic acid under conditions that permit selective hybridization. Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced. Next, the amplification product is detected. In certain methods, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label, or even via a system using electrical or thermal impulse signals.

Methods based on ligation of two (or more) oligonucleotides in the presence of a nucleic acid having the sequence of the resulting “di-oligonucleotide,” thereby amplifying the di-oligonucleotide, also may be used in the amplification step of the disclosure.

In some embodiments of the disclosure, the detection process can utilize a hybridization technique, for example, wherein a specific primer or probe is selected to anneal to a target biomarker of interest, and thereafter detection of selective hybridization is made. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence.

One of skill in the art will recognize that cDNA molecules made using the MMLV RTase mutants of the disclosure can be used in a variety of additional downstream applications. For example, amplification methods may include one-step PCR, two-step PCR, real-time or quantitative PCR, hot-start PCR, nested PCR, touch down PCR, differential display PCR (DDRT-PCR), microarray technologies, inverse PCR, Rapid amplification of PCR ends (RACE or anchored PCR), multiplex PCR, and site directed PCR mutagenesis. Synthesized cDNA and cDNA libraries created with the MMLV RTase mutants of the disclosure can be used in cloning and/or sequencing for further characterization. One of skill in the art will recognize that nucleic acid amplification using cDNA prepared with the MMLV RTase mutants of the disclosure may include additional techniques not listed herein.

To enable hybridization to occur under the methods presented above, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of the sequence of interest.

5. Biological Samples

The MMLV RTase mutants and associated methods of the disclosure may be practiced with any suitable biological sample from which RNA or DNA can be isolated. In one embodiment of the disclosure, the biological sample may be a bodily fluid or tissue obtained from either a diseased or a healthy subject. In some embodiments of the disclosure, the biological sample may be a bodily fluid, including but not limited to whole blood, plasma, serum, feces, or urine. In another embodiment, the methods of the disclosure may be practiced with any suitable samples that are freshly isolated or that have been frozen or stored after having been collected from a subject, for example, with a known diagnosis, treatment, and/or outcome history. Samples may be collected by any non-invasive means, such as, for example, fine needle aspiration or needle biopsy, or alternatively, by an invasive method, including, for example, surgical biopsy. In such embodiments, RNA or DNA can be extracted from a biological sample (e.g., blood serum) before analysis. Methods of RNA and DNA extraction are well known in the art.

A number of kits for use in extracting RNA (i.e., total RNA or mRNA) from bodily fluids or tissues (e.g., blood serum) and are known in the art and commercially available. One of ordinary skill in the art can easily select an appropriate kit for a particular situation.

In certain embodiments of the disclosure, after extraction, mRNA is amplified, and transcribed into cDNA, which can then serve as template for multiple rounds of transcription by the appropriate RNA polymerase. Amplification methods that may be used to practice the methods of the disclosure are described herein and are well known in the art. Reverse transcription reactions may be carried out using non-specific primers, such as an anchored oligo-dT primer, or random sequence primers, or using a target-specific primer complementary to the RNA for each genetic probe being monitored, or using thermostable DNA polymerases, such as MMLV RTase or the MMLV RTase mutants of the disclosure.

In certain embodiments, the RNA isolated from a biological sample (e.g., after amplification and/or conversion to cDNA or cRNA) is labeled with a detectable agent before being analyzed. The role of a detectable agent is to facilitate detection of RNA or to allow visualization of hybridized nucleic acid fragments (e.g., nucleic acid fragments hybridized to genetic probes in an array-based assay). In some embodiments, the detectable agent is selected such that it generates a signal which can be measured and whose intensity is related to the amount of labeled nucleic acids present in the sample being analyzed.

Methods for labeling nucleic acid molecules are well known in the art. A review of labeling protocols and label detection techniques can be found in Kricka, Ann. Clin. Biochem. 39: 114-29 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn. 1: 81-91 (2001); and Joos et al., J. Biotechnol. 35: 135-53 (1994). Standard nucleic acid labeling methods include incorporation of radioactive agents; direct attachment of fluorescent dyes or of enzymes; chemical modifications of nucleic acid fragments making them detectable immunochemically or by other affinity reactions; and enzyme-mediated labeling methods, such as random priming, nick translation, PCR, and tailing with terminal transferase.

Any of a wide variety of detectable agents can be used to practice the methods of the disclosure. Suitable detectable agents include but are not limited to various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, and phosphors), enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, and alkaline phosphatase), colorimetric labels, magnetic labels, biotin, digoxigenin, or other haptens and proteins for which antisera or monoclonal antibodies are available.

6. Kits

The disclosure also provides kits for use in reverse transcription or related technologies. These kits include one or more of the following: an MMLV RTase mutant enzyme, reagents and buffers for conducting a reverse transcriptase reaction, a box, vial tubes, ampules, and the like. Kits can also include instructions for use of the kit for practicing any of the methods disclosed herein or other methods known to those of skill in the art.

EXAMPLES

The claimed invention is further illustrated by the following Examples, which should not be construed as limiting. Those of skill in the art will recognize that the claimed invention may be practiced with variations of the disclosed structures, materials, compositions, and methods, and such variations are regarded as within the scope of the claimed invention.

The RTases described herein were overexpressed in E. coli, purified to homogeneity, and tested for their ability to enhance RNA detection in the context of reverse transcriptase quantitative PCR (RT-qPCR).

Example 1. Preparation of Reverse Transcriptase Mutants by Site Directed Mutagenesis

a. Cloning of MMLV RTase Mutants Created from Base Construct (RNase H Minus Construct)

MM4LV RTase mutants were prepared by first introducing three mutations (D524G, E562Q, and D583N) into the amino acid sequence of the wild-type, or naturally occurring, MMLV RTase to prepare an MMLV RTase base construct (SEQ TD NO: 637). The three mutations, which are contained in the SuperScript II RTase (Invitrogen), have been shown to reduce RNase H activity (see U.S. Pat. No. 5,405,776). The MMLV RTase base construct was optimized for E. coli expression and obtained as gBlocks® Gene Fragments (Integrated DNA Technologies) or by custom gene synthesis with the appropriate purification tag. Subsequent genes were amplified using standard PCR conditions and primers (see Tables 1 and 21). Amplified DNA was subjected to purification using a QIAquick PCR Purification kit (Qiagen, Catalog #28104), followed by gene fragment assembly into a pET28b expression plasmid. Plasmid DNA was isolated and sequenced to verify the desired sequence following transformation into E. coli cells. MMLV RTase mutations were selected by rational design (FIGS. 1A-1C) and introduced by site-directed mutagenesis, using standard PCR conditions and primers (see Tables 1 and 21). Resulting plasmids were transformed into E. coli BL21(DE3) cells for expression.

TABLE 1 Sequences of primers used for cloning of MMLV RTase base constructs and mutants into pET28b. SEQ ID Primer Sequence NO: Primer Name (5′-3′) 1 pET28b 5′ GGTATATCTCCTTCT Reverse TAAAGTTAAACAAAA TTATTTCTAGAGGGG AAT 2 pET28b 3′ GATCCGGCTGCTAAC Forward AAAGCC 3 MMLV 5′ Primer TTTTGTTTAACTTTA AGAAGGAGATATACC ATGGGCAGCAGCCAT CATCATC 4 MMLV 3′ Primer GCAGCCAACTCAGCT TCCTTTCGGGCTTTG TTAAAAATGCTCGCT AGTGTAGGGAGAGC 5 MMLV K53A Top AAGCACCGTTGATCA SDM TCCCGTTAGCGGCAA CGTCTACACCTGTCT CTATCAAAC 6 MMLV K53R Top AAGCACCGTTGATCA SDM TCCCGTTACGTGCAA CGTCTACACCTGTCT CTATCAAAC 7 MMLV K53E Top AAGCACCGTTGATCA SDM TCCCGTTAGAAGCAA CGTCTACACCTGTCT CTATCAAAC 8 MMLV T55A Top CCGTTGATCATCCCG SDM TTAAAGGCAGCGTCT ACACCTGTCTCTATC AAACAGTACCCC 9 MMLV T55R Top CCGTTGATCATCCCG SDM TTAAAGGCACGTTCT ACACCTGTCTCTATC AAACAGTACCCC 10 MMLV T55E Top CCGTTGATCATCCCG SDM TTAAAGGCAGAATCT ACACCTGTCTCTATC AAACAGTACCCC 11 MMLV T57A Top ATCATCCCGTTAAAG SDM GCAACGTCTGCGCCT GTCTCTATCAAACAG TACCCCATGAG 12 MMLV T57R Top ATCATCCCGTTAAAG SDM GCAACGTCTCGTCCT GTCTCTATCAAACAG TACCCCATGAG 13 MMLV T57E Top ATCATCCCGTTAAAG SDM GCAACGTCTGAACCT GTCTCTATCAAACAG TACCCCATGAG 14 MMLV V59A Top CCGTTAAAGGCAACG SDM TCTACACCTGCGTCT ATCAAACAGTACCCC ATGAGTCAAGAGG 15 MMLV V59R Top CCGTTAAAGGCAACG SDM TCTACACCTCGTTCT ATCAAACAGTACCCC ATGAGTCAAGAGG 16 MMLV V59E Top CCGTTAAAGGCAACG SDM TCTACACCTGAATCT ATCAAACAGTACCCC ATGAGTCAAGAGG 17 MMLV 161A Top TAAAGGCAACGTCTA SDM CACCTGTCTCTGCGA AACAGTACCCCATGA GTCAAGAGG 18 MMLV I61R Top TAAAGGCAACGTCTA SDM CACCTGTCTCTCGTA AACAGTACCCCATGA GTCAAGAGG 19 MMLV 16IE Top TAAAGGCAACGTCTA SDM CACCTGTCTCTGAAA AACAGTACCCCATGA GTCAAGAGG 20 MMLV K62A Top GGCAACGTCTACACC SDM TGTCTCTATCGCGCA GTACCCCATGAGTCA AGAGGC 21 MMLV K62R Top GGCAACGTCTACACC SDM TGTCTCTATCCGTCA GTACCCCATGAGTCA AGAGGC 22 MMLV K62E Top GGCAACGTCTACACC SDM TGTCTCTATCGAACA GTACCCCATGAGTCA AGAGGC 23 MMLV Q68A Top CTGTCTCTATCAAAC SDM AGTACCCCATGAGTG CGGAGGCCCGCCTGG G 24 MMLV Q68R Top CTGTCTCTATCAAAC SDM AGTACCCCATGAGTC GTGAGGCCCGCCTGG G 25 MMLV Q68E Top CTGTCTCTATCAAAC SDM AGTACCCCATGAGTG AAGAGGCCCGCCTGG G 26 MMLV K75A Top GGCCCGCCTGGGGAT SDM TGCGCCACATATTCA GCGCTTGCTGGACCA 27 MMLV K75R Top GGCCCGCCTGGGGAT SDM TCGTCCACATATTCA GCGCTTGCTGGACCA 28 MMLV K75E Top GGCCCGCCTGGGGAT SDM TGAACCACATATTCA GCGCTTGCTGGACCA 29 MMLV Q79A Top CGCCTGGGGATTAAG SDM CCACATATTGCGCGC TTGCTGGACCAGGGG 30 MMLV Q79R Top CGCCTGGGGATTAAG SDM CCACATATTCGTCGC TTGCTGGACCAGGGG 31 MMLV Q79E Top CGCCTGGGGATTAAG SDM CCACATATTGAACGC TTGCTGGACCAGGGG 32 MMLV L99A Top CCGTGGAACACCCCC SDM CTTGCGCCCGTGAAA AAGCCAGGTACAAAC 33 MMLV L99R Top CCGTGGAACACCCCC SDM CTTCGTCCCGTGAAA AAGCCAGGTACAAAC 34 MMLV L99E Top CCGTGGAACACCCCC SDM CTTGAACCCGTGAAA AAGCCAGGTACAAAC 35 MMLV V101A Top CACCCCCCTTCTGCC SDM CGCGAAAAAGCCAGG TACAAACGATTATCG TCC 36 MMLV V101R Top CACCCCCCTTCTGCC SDM CCGTAAAAAGCCAGG TACAAACGATTATCG TCC 37 MMLV V101E Top CACCCCCCTTCTGCC SDM CGAAAAAAAGCCAGG TACAAACGATTATCG TCC 38 MMLV K102A Top CCCCCTTCTGCCCGT SDM GGCGAAGCCAGGTAC AAACGATTATCGTCC 39 MMLV K102R Top CCCCCTTCTGCCCGT SDM GCGTAAGCCAGGTAC AAACGATTATCGTCC 40 MMLV K102E Top CCCCCTTCTGCCCGT SDM GGAAAAGCCAGGTAC AAACGATTATCGTCC 41 MMLV K103A Top CCCCCTTCTGCCCGT SDM GAAAGCGCCAGGTAC AAACGATTATCGTCC AGTT 42 MMLV K103R Top CCCCCTTCTGCCCGT SDM GAAACGTCCAGGTAC AAACGATTATCGTCC AGTT 43 MMLV K103E Top CCCCCTTCTGCCCGT SDM GAAAGAACCAGGTAC AAACGATTATCGTCC AGTT 44 MMLV T106A Top GCCCGTGAAAAAGCC SDM AGGTGCGAACGATTA TCGTCCAGTTCAAGA TCTTCG 45 MMLV T106R Top GCCCGTGAAAAAGCC SDM AGGTCGTAACGATTA TCGTCCAGTTCAAGA TCTTCG 46 MMLV T106E Top GCCCGTGAAAAAGCC SDM AGGTGAAAACGATTA TCGTCCAGTTCAAGA TCTTCG 47 MMLV N107A Top CCCGTGAAAAAGCCA SDM GGTACAGCGGATTAT CGTCCAGTTCAAGAT CTTCGCG 48 MMLV N107R Top CCCGTGAAAAAGCCA SDM GGTACACGTGATTAT CGTCCAGTTCAAGAT CTTCGCG 49 MMLV N107E CCCGTGAAAAAGCCAGGTAC Top SDM AGAAGATTATCGTCCAGTTC AAGATCTTCGCG 50 MMLV Y109A CGTGAAAAAGCCAGGTACAA Top SDM ACGATGCGCGTCCAGTTCAA GATCTTCGCG 51 MMLV Y109R CGTGAAAAAGCCAGGTACAA Top SDM ACGATCGTCGTCCAGTTCAA GATCTTCGCG 52 MMLV Y109E CGTGAAAAAGCCAGGTACAA Top SDM ACGATGAACGTCCAGTTCAA GATCTTCGCG 53 MMLVR110A CGTGAAAAAGCCAGGTACAA Top SDM ACGATTATGCGCCAGTTCAA GATCTTCGCGAGG 54 MMLVR110K CGTGAAAAAGCCAGGTACAA Top SDM ACGATTATAAACCAGTTCAA GATCTTCGCGAGG 55 MMLV R110E CGTGAAAAAGCCAGGTACAA Top SDM ACGATTATGAACCAGTTCAA GATCTTCGCGAGG 56 MMLV V112A GCCAGGTACAAACGATTATC Top SDM GTCCAGCGCAAGATCTTCGC GAGGTCAACAAAC 57 MMLV VI12R GCCAGGTACAAACGATTATC Top SDM GTCCACGTCAAGATCTTCGC GAGGTCAACAAAC 58 MMLV V112E GCCAGGTACAAACGATTATC Top SDM GTCCAGAACAAGATCTTCGC GAGGTCAACAAAC 59 MMLV K120A AGTTCAAGATCTTCGCGAGG Top SDM TCAACGCGCGCGTAGAAGAC ATCCATCCGAC 60 MMLV K120R AGTTCAAGATCTTCGCGAGG Top SDM TCAACCGTCGCGTAGAAGAC ATCCATCCGAC 61 MMLV K120E AGTTCAAGATCTTCGCGAGG Top SDM TCAACGAACGCGTAGAAGAC ATCCATCCGAC 62 MMLV El23A GCGAGGTCAACAAACGCGTA Top SDM GCGGACATCCATCCGACTGT ACCTAATCC 63 MMLV E123R GCGAGGTCAACAAACGCGTA Top SDM CGTGACATCCATCCGACTGT ACCTAATCC 64 MMLV E123D GCGAGGTCAACAAACGCGTA Top SDM GATGACATCCATCCGACTGT ACCTAATCC 65 MMLV T128V ACGCGTAGAAGACATCCATC Top SDM CGGTGGTACCTAATCCTTAT AATCTGTTATCAGGCCTGC 66 MMLV T128R ACGCGTAGAAGACATCCATC Top SDM CGCGTGTACCTAATCCTTAT AATCTGTTATCAGGCCTGC 67 MMLV T128E ACGCGTAGAAGACATCCATC Top SDM CGGAAGTACCTAATCCTTAT AATCTGTTATCAGGCCTGC 68 MMLV K193A CGTCTGCCCCAGGGCTTTGC Top SDM GAACAGCCCCACATTGTTCG ATGAA 69 MMLV K193R CGTCTGCCCCAGGGCTTTCG Top SDM TAACAGCCCCACATTGTTCG ATGAA 70 MMLV K193E CGTCTGCCCCAGGGCTTTGA Top SDM AAACAGCCCCACATTGTTCG ATGAA 71 MMLV E282A AGAAGGTCAACGTTGGCTGA Top SDM CTGCGGCGCGTAAGGAGACC GTAATG 72 MMLV E282R AGAAGGTCAACGTTGGCTGA Top SDM CTCGTGCGCGTAAGGAGACC GTAATG 73 MMLV E282D AGAAGGTCAACGTTGGCTGA Top SDM CTGATGCGCGTAAGGAGACC GTAATG 74 MMLV A283V GAAGGTCAACGTTGGCTGAC Top SDM TGAAGTGCGTAAGGAGACCG TAATGGGGC 75 MMLV A283R GAAGGTCAACGTTGGCTGAC Top SDM TGAACGTCGTAAGGAGACCG TAATGGGGC 76 MMLV A283E GAAGGTCAACGTTGGCTGAC Top SDM TGAAGAACGTAAGGAGACCG TAATGGGGC 77 MMLV Q291A GCGTAAGGAGACCGTAATGG Top SDM GGGCGCCTACGCCTAAGACG CCACG 78 MMLV Q291R GCGTAAGGAGACCGTAATGG Top SDM GGCGTCCTACGCCTAAGACG CCACG 79 MMLV Q291E GCGTAAGGAGACCGTAATGG Top SDM GGGAACCTACGCCTAAGACG CCACG 80 MMLV GAGACCGTAATGGGGCAGCC T293A TGCGCCTAAGACGCCACGCC Top SDM AGTTG 81 MMLV GAGACCGTAATGGGGCAGCC T293R TCGTCCTAAGACGCCACGCC Top SDM AGTTG 82 MMLV GAGACCGTAATGGGGCAGCC T293E TGAACCTAAGACGCCACGCC Top SDM AGTTG 83 MMLV K295A GTAATGGGGCAGCCTACGCC Top SDM TGCGACGCCACGCCAGTTGC GTGAA 84 MMLV K295R GTAATGGGGCAGCCTACGCC Top SDM TCGTACGCCACGCCAGTTGC GTGAA 85 MMLV K295E GTAATGGGGCAGCCTACGCC Top SDM TGAAACGCCACGCCAGTTGC GTGAA 86 MMLV TGGGGCAGCCTACGCCTAAG T296A GCGCCACGCCAGTTGCGTGA Top SDM ATTTT 87 MMLV TGGGGCAGCCTACGCCTAAG T296R CGTCCACGCCAGTTGCGTGA Top SDM ATTTT 88 MMLV TGGGGCAGCCTACGCCTAAG T296E GAACCACGCCAGTTGCGTGA Top SDM ATTTT 89 MMLV R298A GCCTACGCCTAAGACGCCAG Top SDM CGCAGTTGCGTGAATTTTTG GGCACAG 90 MMLV R298K GCCTACGCCTAAGACGCCAA Top SDM AACAGTTGCGTGAATTTTTG GGCACAG 91 MMLV R298E GCCTACGCCTAAGACGCCAG Top SDM AACAGTTGCGTGAATTTTTG GGCACAG 92 MMLV R30IA CCTAAGACGCCACGCCAGTT Top SDM GGCGGAATTTTTGGGCACAG CGGGA 93 MMLV R301K CCTAAGACGCCACGCCAGTT Top SDM GAAAGAATTTTTGGGCACAG CGGGA 94 MMLV R301E CCTAAGACGCCACGCCAGTT Top SDM GGAAGAATTTTTGGGCACAG CGGGA 95 MMLV K329A GCACCCCTGTACCCCTTAAC Top SDM AGCGACAGGGACGCTTTTCA ACTGG 96 MMLV K329R GCACCCCTGTACCCCTTAAC Top SDM ACGTACAGGGACGCTTTTCA ACTGG 97 MMLV K329E GCACCCCTGTACCCCTTAAC Top SDM AGAAACAGGGACGCTTTTCA ACTGG 98 MMLV K53A GTTTGATAGAGACAGGTGTA Btm SDM GACGTTGCCGCTAACGGGAT GATCAACGGTGCTT 99 MMLV K53R GTTTGATAGAGACAGGTGTA Btm SDM GACGTTGCACGTAACGGGAT GATCAACGGTGCTT 100 MMLV K53E GTTTGATAGAGACAGGTGTA Btm SDM GACGTTGCTTCTAACGGGAT GATCAACGGTGCTT 101 MMLV GGGGTACTGTTTGATAGAGA T55A CAGGTGTAGACGCTGCCTTT Btm SDM AACGGGATGATCAACGG 102 MMLV GGGGTACTGTTTGATAGAGA T55R CAGGTGTAGAACGTGCCTTT Btm SDM AACGGGATGATCAACGG 103 MMLV GGGGTACTGTTTGATAGAGA T55E CAGGTGTAGATTCTGCCTTT Btm SDM AACGGGATGATCAACGG 104 MMLV CTCATGGGGTACTGTTTGAT T57A AGAGACAGGCGCAGACGTTG Btm SDM CCTTTAACGGGATGAT 105 MMLV CTCATGGGGTACTGTTTGAT T57R AGAGACAGGACGAGACGTTG Btm SDM CCTTTAACGGGATGAT 106 MMLV CTCATGGGGTACTGTTTGAT T57E AGAGACAGGTTCAGACGTTG Btm SDM CCTTTAACGGGATGAT 107 MMLV V59A CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGATAGACGCAGGTGT AGACGTTGCCTTTAACGG 108 MMLV V59R CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGATAGAACGAGGTGT AGACGTTGCCTTTAACGG 109 MMLV V59E CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGATAGATTCAGGTGT AGACGTTGCCTTTAACGG 110 MMLV 161A CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCGCAGAGACAGGTGT AGACGTTGCCTTTA 111 MMLV 161R CCTCTTGACTCATGGGGTAC Btm SDM TGTTTACGAGAGACAGGTGT AGACGTTGCCTTTA 112 MMLV 16IE CCTCTTGACTCATGGGGTAC Btm SDM TGTTTTTCAGAGACAGGTGT AGACGTTGCCTTTA 113 MMLV K62A GCCTCTTGACTCATGGGGTA Btm SDM CTGCGCGATAGAGACAGGTG TAGACGTTGCC 114 MMLV K62R GCCTCTTGACTCATGGGGTA Btm SDM CTGACGGATAGAGACAGGTG TAGACGTTGCC 115 MMLV K62E GCCTCTTGACTCATGGGGTA Btm SDM CTGTTCGATAGAGACAGGTG TAGACGTTGCC 116 MMLV Q68A CTGTCTCTATCAAACAGTAC Btm SDM CCCATGAGTGCGGAGGCCCG CCTGGG 117 MMLV Q68R CTGTCTCTATCAAACAGTAC Btm SDM CCCATGAGTCGTGAGGCCCG CCTGGG 118 MMLV Q68E CTGTCTCTATCAAACAGTAC Btm SDM CCCATGAGTGAAGAGGCCCG CCTGGG 119 MMLV K75A TGGTCCAGCAAGCGCTGAAT Btm SDM ATGTGGCGCAATCCCCAGGC GGGCC 120 MMLV K75R TGGTCCAGCAAGCGCTGAAT Btm SDM ATGTGGACGAATCCCCAGGC GGGCC 121 MMLV K75E TGGTCCAGCAAGCGCTGAAT Btm SDM ATGTGGTTCAATCCCCAGGC GGGCC 122 MMLV Q79A CCCCTGGTCCAGCAAGCGCG Btm SDM CAATATGTGGCTTAATCCCC AGGCG 123 MMLV Q79R CCCCTGGTCCAGCAAGCGAC Btm SDM GAATATGTGGCTTAATCCCC AGGCG 124 MMLV Q79E CCCCTGGTCCAGCAAGCGTT Btm SDM CAATATGTGGCTTAATCCCC AGGCG 125 MMLV L99A GTTTGTACCTGGCTTTTTCA Btm SDM CGGGCGCAAGGGGGGTGTTC CACGG 126 MMLV L99R GTTTGTACCTGGCTTTTTCA Btm SDM CGGGACGAAGGGGGGTGTTC CACGG 127 MMLV L99E GTTTGTACCTGGCTTTTTCA Btm SDM CGGGTTCAAGGGGGGTGTTC CACGG 128 MMLV V101A GGACGATAATCGTTTGTACC Btm SDM TGGCTTTTTCGCGGGCAGAA GGGGGGTG 129 MMLV VI01R GGACGATAATCGTTTGTACC Btm SDM TGGCTTTTTACGGGGCAGAA GGGGGGTG 130 MMLV V101E GGACGATAATCGTTTGTACC Btm SDM TGGCTTTTTTTCGGGCAGAA GGGGGGTG 131 MMLV K102A GGACGATAATCGTTTGTACC Btm SDM TGGCTTCGCCACGGGCAGAA GGGGG 132 MMLV K102R GGACGATAATCGTTTGTACC Btm SDM TGGCTTACGCACGGGCAGAA GGGGG 133 MMLV K102E GGACGATAATCGTTTGTACC Btm SDM TGGCTTTTCCACGGGCAGAA GGGGG 134 MMLV K103 A AACTGGACGATAATCGTTTG Btm SDM TACCTGGCGCTTTCACGGGC AGAAGGGGG 135 MMLV K103R AACTGGACGATAATCGTTTG Btm SDM TACCTGGACGTTTCACGGGC AGAAGGGGG 136 MMLV K103E AACTGGACGATAATCGTTTG Btm SDM TACCTGGTTCTTTCACGGGC AGAAGGGGG 137 MMLV CGAAGATCTTGAACTGGACG T106A ATAATCGTTCGCACCTGGCT Btm SDM TTTTCACGGGC 138 MMLV CGAAGATCTTGAACTGGACG T106R ATAATCGTTACGACCTGGCT Btm SDM TTTTCACGGGC 139 MMLV CGAAGATCTTGAACTGGACG T106E ATAATCGTTTTCACCTGGCT Btm SDM TTTTCACGGGC 140 MMLVN107A CGCGAAGATCTTGAACTGGA Btm SDM CGATAATCCGCTGTACCTGG CTTTTTCACGGG 141 MMLV N107R CGCGAAGATCTTGAACTGGA Btm SDM CGATAATCACGTGTACCTGG CTTTTTCACGGG 142 MMLV N107E CGCGAAGATCTTGAACTGGA Btm SDM CGATAATCTTCTGTACCTGG CTTTTTCACGGG 143 MMLV Y109A CGCGAAGATCTTGAACTGGA Btm SDM CGCGCATCGTTTGTACCTGG CTTTTTCACG 144 MMLV Y109R CGCGAAGATCTTGAACTGGA Btm SDM CGACGATCGTTTGTACCTGG CTTTTTCACG 145 MMLV Y109E CGCGAAGATCTTGAACTGGA Btm SDM CGTTCATCGTTTGTACCTGG CTTTTTCACG 146 MMLVR110A CCTCGCGAAGATCTTGAACT Btm SDM GGCGCATAATCGTTTGTACC TGGCTTTTTCACG 147 MMLV R110K CCTCGCGAAGATCTTGAACT Btm SDM GGTTTATAATCGTTTGTACC TGGCTTTTTCACG 148 MMLVR110E CCTCGCGAAGATCTTGAACT Btm SDM GGTTCATAATCGTTTGTACC TGGCTTTTTCACG 149 MMLV V112A GTTTGTTGACCTCGCGAAGA Btm SDM TCTTGCGCTGGACGATAATC GTTTGTACCTGGC 150 MMLV V112R GTTTGTTGACCTCGCGAAGA Btm SDM TCTTGACGTGGACGATAATC GTTTGTACCTGGC 151 MMLV VI12E GTTTGTTGACCTCGCGAAGA Btm SDM TCTTGTTCTGGACGATAATC GTTTGTACCTGGC 152 MMLV K120A GTCGGATGGATGTCTTCTAC Btm SDM GCGCGCGTTGACCTCGCGAA GATCTTGAACT 153 MMLV K120R GTCGGATGGATGTCTTCTAC Btm SDM GCGACGGTTGACCTCGCGAA GATCTTGAACT 154 MMLV K120E GTCGGATGGATGTCTTCTAC Btm SDM GCGTTCGTTGACCTCGCGAA GATCTTGAACT 155 MMLV E123A GGATTAGGTACAGTCGGATG Btm SDM GATGTCCGCTACGCGTTTGT TGACCTCGC 156 MMLV E123R GGATTAGGTACAGTCGGATG Btm SDM GATGTCACGTACGCGTTTGT TGACCTCGC 157 MMLV E123D GGATTAGGTACAGTCGGATG Btm SDM GATGTCATCTACGCGTTTGT TGACCTCGC 158 MMLV GCAGGCCTGATAACAGATTA T128V TAAGGATTAGGTACCACCGG Btm SDM ATGGATGTCTTCTACGCGT 159 MMLV GCAGGCCTGATAACAGATTA T128R TAAGGATTAGGTACACGCGG Btm SDM ATGGATGTCTTCTACGCGT 160 MMLV GCAGGCCTGATAACAGATTA T128E TAAGGATTAGGTACTTCCGG Btm SDM ATGGATGTCTTCTACGCGT 161 MMLV K193A TTCATCGAACAATGTGGGGC Btm SDM TGTTCGCAAAGCCCTGGGGC AGACG 162 MMLV K193R TTCATCGAACAATGTGGGGC Btm SDM TGTTACGAAAGCCCTGGGGC AGACG 163 MMLV K193E TTCATCGAACAATGTGGGGC Btm SDM TGTTTTCAAAGCCCTGGGGC AGACG 164 MMLV E282A CATTACGGTCTCCTTACGCG Btm SDM CCGCAGTCAGCCAACGTTGA CCTTCT 165 MMLV E282R CATTACGGTCTCCTTACGCG Btm SDM CACGAGTCAGCCAACGTTGA CCTTCT 166 MMLV E282D CATTACGGTCTCCTTACGCG Btm SDM CATCAGTCAGCCAACGTTGA CCTTCT 167 MMLV A283V GCCCCATTACGGTCTCCTTA Btm SDM CGCACTTCAGTCAGCCAACG TTGACCTTC 168 MMLV A283R GCCCCATTACGGTCTCCTTA Btm SDM CGACGTTCAGTCAGCCAACG TTGACCTTC 169 MMLV A283E GCCCCATTACGGTCTCCTTA Btm SDM CGTTCTTCAGTCAGCCAACG TTGACCTTC 170 MMLV Q29IA CGTGGCGTCTTAGGCGTAGG Btm SDM CGCCCCCATTACGGTCTCCT TACGC 171 MMLV Q291R CGTGGCGTCTTAGGCGTAGG Btm SDM ACGCCCCATTACGGTCTCCT TACGC 172 MMLV Q291E CGTGGCGTCTTAGGCGTAGG Btm SDM TTCCCCCATTACGGTCTCCT TACGC 173 MMLV T293A CAACTGGCGTGGCGTCTTAG Btm SDM GCGCAGGCTGCCCCATTACG GTCTC 174 MMLV T293R CAACTGGCGTGGCGTCTTAG Btm SDM GACGAGGCTGCCCCATTACG GTCTC 175 MMLV T293E CAACTGGCGTGGCGTCTTAG Btm SDM GTTCAGGCTGCCCCATTACG GTCTC 176 MMLV K295A TTCACGCAACTGGCGTGGCG Btm SDM TCGCAGGCGTAGGCTGCCCC ATTAC 177 MMLV K295R TTCACGCAACTGGCGTGGCG Btm SDM TACGAGGCGTAGGCTGCCCC ATTAC 178 MMLV K295E TTCACGCAACTGGCGTGGCG Btm SDM TTTCAGGCGTAGGCTGCCCC ATTAC 179 MMLV T296A AAAATTCACGCAACTGGCGT Btm SDM GGCGCCTTAGGCGTAGGCTG CCCCA 180 MMLV T296R AAAATTCACGCAACTGGCGT Btm SDM GGACGCTTAGGCGTAGGCTG CCCCA 181 MMLV T296E AAAATTCACGCAACTGGCGT Btm SDM GGTTCCTTAGGCGTAGGCTG CCCCA 182 MMLV R298A CTGTGCCCAAAAATTCACGC Btm SDM AACTGCGCTGGCGTCTTAGG CGTAGGC 183 MMLV R298K CTGTGCCCAAAAATTCACGC Btm SDM AACTGTTTTGGCGTCTTAGG CGTAGGC 184 MMLV R298E CTGTGCCCAAAAATTCACGC Btm SDM AACTGTTCTGGCGTCTTAGG CGTAGGC 185 MMLV R301A TCCCGCTGTGCCCAAAAATT Btm SDM CCGCCAACTGGCGTGGCGTC TTAGG 186 MMLV R301K TCCCGCTGTGCCCAAAAATT Btm SDM CTTTCAACTGGCGTGGCGTC TTAGG 187 MMLV R301E TCCCGCTGTGCCCAAAAATT Btm SDM CTTCCAACTGGCGTGGCGTC TTAGG 188 MMLV K329A CCAGTTGAAAAGCGTCCCTG Btm SDM TCGCTGTTAAGGGGTACAGG GGTGC 189 MMLV K329R CCAGTTGAAAAGCGTCCCTG Btm SDM TACGTGTTAAGGGGTACAGG GGTGC 190 MMLV K329E CCAGTTGAAAAGCGTCCCTG Btm SDM TTTCTGTTAAGGGGTACAGG GGTGC 191 MMLV I61G TAAAGGCAACGTCTACACCT Top SDM GTCTCTGGCAAACAGTACCC CATGAGTCAAGAGG 192 MMLV 161G CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGCCAGAGACAGGTGT AGACGTTGCCTTTA 193 MMLV 161L TAAAGGCAACGTCTACACCT Top SDM GTCTCTCTGAAACAGTACCC CATGAGTCAAGAGG 194 MMLV 161L CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCAGAGAGACAGGTGT AGACGTTGCCTTTA 195 MMLV 16IV TAAAGGCAACGTCTACACCT Top SDM GTCTCTGTGAAACAGTACCC CATGAGTCAAGAGG 196 MMLV 16IV CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCACAGAGACAGGTGT AGACGTTGCCTTTA 197 MMLV 16IP TAAAGGCAACGTCTACACCT Top SDM GTCTCTCCGAAACAGTACCC CATGAGTCAAGAGG 198 MMLV 16IP CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCGGAGAGACAGGTGT AGACGTTGCCTTTA 199 MMLV 161M TAAAGGCAACGTCTACACCT Top SDM GTCTCTATGAAACAGTACCC CATGAGTCAAGAGG 200 MMLV 161M CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCATAGAGACAGGTGT AGACGTTGCCTTTA 201 MMLV 16IS TAAAGGCAACGTCTACACCT Top SDM GTCTCTAGCAAACAGTACCC CATGAGTCAAGAGG 202 MMLV 16IS CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGCTAGAGACAGGTGT AGACGTTGCCTTTA 203 MMLV 16IT TAAAGGCAACGTCTACACCT Top SDM GTCTCTACCAAACAGTACCC CATGAGTCAAGAGG 204 MMLV 16IT CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGGTAGAGACAGGTGT AGACGTTGCCTTTA 205 MMLV 161C TAAAGGCAACGTCTACACCT Top SDM GTCTCTTGCAAACAGTACCC CATGAGTCAAGAGG 206 MMLV 161C CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGCAAGAGACAGGTGT AGACGTTGCCTTTA 207 MMLV 16IF TAAAGGCAACGTCTACACCT Top SDM GTCTCTTTTAAACAGTACCC CATGAGTCAAGAGG 208 MMLV 161F CCTCTTGACTCATGGGGTAC Btm TGTTTAAAAGAGACA SDM GGTGTAGACGTTGCCTTTA 209 MMLV 161Y TAAAGGCAACGTCTACACCT Top SDM GTCTCTTATAAACAGTACCC CATGAGTCAAGAGG 210 MMLV 161Y CCTCTTGACTCATGGGGTAC Btm SDM TGTTTATAAGAGACAGGTGT AGACGTTGCCTTTA 211 MMLV 161H TAAAGGCAACGTCTACACCT Top SDM GTCTCTCATAAACAGTACCC CATGAGTCAAGAGG 212 MMLV 161H CCTCTTGACTCATGGGGTAC Btm SDM TGTTTATGAGAGACAGGTGT AGACGTTGCCTTTA 213 MMLV I61W TAAAGGCAACGTCTACACCT Top SDM GTCTCTTGGAAACAGTACCC CATGAGTCAAGAGG 214 MMLV 161W CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCCAAGAGACAGGTGT AGACGTTGCCTTTA 215 MMLV 161D TAAAGGCAACGTCTACACCT Top SDM GTCTCTGATAAACAGTACCC CATGAGTCAAGAGG 216 MMLV 16ID CCTCTTGACTCATGGGGTAC Btm SDM TGTTTATCAGAGACAGGTGT AGACGTTGCCTTTA 217 MMLV 16IN TAAAGGCAACGTCTACACCT Top SDM GTCTCTAACAAACAGTACCC CATGAGTCAAGAGG 218 MMLV 16IN CCTCTTGACTCATGGGGTAC Btm SDM TGTTTGTTAGAGACAGGTGT AGACGTTGCCTTTA 219 MMLV 16IQ TAAAGGCAACGTCTACACCT Top SDM GTCTCTCAGAAACAGTACCC CATGAGTCAAGAGG 220 MMLV 16IQ CCTCTTGACTCATGGGGTAC Btm SDM TGTTTCTGAGAGACAGGTGT AGACGTTGCCTTTA 221 MMLV 161K TAAAGGCAACGTCTACACCT Top SDM GTCTCTAAAAAACAGTACCC CATGAGTCAAGAGG 222 MMLV 16IK CCTCTTGACTCATGGGGTAC Btm SDM TGTTTTTTAGAGACAGGTGT AGACGTTGCCTTTA 223 MMLV Q68G CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTGGCGAGGCCCG CCTGGG 224 MMLV Q68G CCCAGGCGGGCCTCGCCACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 225 MMLV Q68L CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTCTGGAGGCCCG CCTGGG 226 MMLV Q68L CCCAGGCGGGCCTCCAGACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 227 MMLV Q68I CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTATTGAGGCCCG CCTGGG 228 MMLV Q68I CCCAGGCGGGCCTCAATACT Btm CATGGGGTACTGTTT SDM GATAGAGACAG 229 MMLV Q68V CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTGTGGAGGCCCG CCTGGG 230 MMLV Q68V CCCAGGCGGGCCTCCACACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 231 MMLV Q68P CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTCCGGAGGCCCG CCTGGG 232 MMLV Q68P CCCAGGCGGGCCTCCGGACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 233 MMLV Q68M CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTATGGAGGCCCG CCTGGG 234 MMLV Q68M CCCAGGCGGGCCTCCATACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 235 MMLV Q68S CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTAGCGAGGCCCG CCTGGG 236 MMLV Q68S CCCAGGCGGGCCTCGCTACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 237 MMLV Q68T CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTACCGAGGCCCG CCTGGG 238 MMLV Q68T CCCAGGCGGGCCTCGGTACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 239 MMLV Q68C CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTTGCGAGGCCCG CCTGGG 240 MMLV Q68C CCCAGGCGGGCCTCGCAACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 241 MMLV Q68F CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTTTTGAGGCCCG CCTGGG 242 MMLV Q68F CCCAGGCGGGCCTCAAAACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 243 MMLV Q68Y CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTTATGAGGCCCG CCTGGG 244 MMLV Q68Y CCCAGGCGGGCCTCATAACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 245 MMLV Q68H CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTCATGAGGCCCG CCTGGG 246 MMLV Q68H CCCAGGCGGGCCTCATGACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 247 MMLV Q68W CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTTGGGAGGCCCG CCTGGG 248 MMLV Q68W CCCAGGCGGGCCTCCCAACT Btm CATGGGGTACTGTTT SDM GATAGAGACAG 249 MMLV Q68D CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTGATGAGGCCCG CCTGGG 250 MMLV Q68D CCCAGGCGGGCCTCATCACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 251 MMLV Q68N CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTAACGAGGCCCG CCTGGG 252 MMLV Q68N CCCAGGCGGGCCTCGTTACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 253 MMLV Q68K CTGTCTCTATCAAACAGTAC Top SDM CCCATGAGTAAAGAGGCCCG CCTGGG 254 MMLV Q68K CCCAGGCGGGCCTCTTTACT Btm SDM CATGGGGTACTGTTTGATAG AGACAG 255 MMLV Q79G CGCCTGGGGATTAAGCCACA Top SDM TATTGGCCGCTTGCTGGACC AGGGG 256 MMLV Q79G CCCCTGGTCCAGCAAGCGGC Btm SDM CAATATGTGGCTTAATCCCC AGGCG 257 MMLV Q79L CGCCTGGGGATTAAGCCACA Top SDM TATTCTGCGCTTGCTGGACC AGGGG 258 MMLV Q79L CCCCTGGTCCAGCAAGCGCA Btm SDM GAATATGTGGCTTAATCCCC AGGCG 259 MMLV Q79I CGCCTGGGGATTAAGCCACA Top SDM TATTATTCGCTTGCTGGACC AGGGG 260 MMLV Q79I CCCCTGGTCCAGCAAGCGAA Btm SDM TAATATGTGGCTTAATCCCC AGGCG 261 MMLV Q79V CGCCTGGGGATTAAGCCACA Top SDM TATTGTGCGCTTGCTGGACC AGGGG 262 MMLV Q79V CCCCTGGTCCAGCAAGCGCA Btm SDM CAATATGTGGCTTAATCCCC AGGCG 263 MMLV Q79P CGCCTGGGGATTAAGCCACA Top SDM TATTCCGCGCTTGCTGGACC AGGGG 264 MMLV Q79P CCCCTGGTCCAGCAAGCGCG Btm SDM GAATATGTGGCTTAATCCCC AGGCG 265 MMLV Q79M CGCCTGGGGATTAAGCCACA Top SDM TATTATGCGCTTGCTGGACC AGGGG 266 MMLV Q79M CCCCTGGTCCAGCAAGCGCA Btm SDM TAATATGTGGCTTAATCCCC AGGCG 267 MMLV Q79S CGCCTGGGGATTAAGCCACA Top SDM TATTAGCCGCTTGCTGGACC AGGGG 268 MMLV Q79S CCCCTGGTCCAGCAAGCGGC Btm TAATATGTGGCTTAA SDM TCCCCAGGCG 269 MMLV Q79T CGCCTGGGGATTAAGCCACA Top SDM TATTACCCGCTTGCTGGACC AGGGG 270 MMLV Q79T CCCCTGGTCCAGCAAGCGGG Btm SDM TAATATGTGGCTTAATCCCC AGGCG 271 MMLV Q79C CGCCTGGGGATTAAGCCACA Top SDM TATTTGCCGCTTGCTGGACC AGGGG 272 MMLV Q79C CCCCTGGTCCAGCAAGCGGC Btm SDM AAATATGTGGCTTAATCCCC AGGCG 273 MMLV Q79F CGCCTGGGGATTAAGCCACA Top SDM TATTTTTCGCTTGCTGGACC AGGGG 274 MMLV Q79F CCCCTGGTCCAGCAAGCGAA Btm SDM AAATATGTGGCTTAATCCCC AGGCG 275 MMLV Q79Y CGCCTGGGGATTAAGCCACA Top SDM TATTTATCGCTTGCTGGACC AGGGG 276 MMLV Q79Y CCCCTGGTCCAGCAAGCGAT Btm SDM AAATATGTGGCTTAATCCCC AGGCG 277 MMLV Q79H CGCCTGGGGATTAAGCCACA Top SDM TATTCATCGCTTGCTGGACC AGGGG 278 MMLV Q79H CCCCTGGTCCAGCAAGCGAT Btm SDM GAATATGTGGCTTAATCCCC AGGCG 279 MMLV Q79W CGCCTGGGGATTAAGCCACA Top SDM TATTTGGCGCTTGCTGGACC AGGGG 280 MMLV Q79W CCCCTGGTCCAGCAAGCGCC Btm SDM AAATATGTGGCTTAATCCCC AGGCG 281 MMLV Q79D CGCCTGGGGATTAAGCCACA Top SDM TATTGATCGCTTGCTGGACC AGGGG 282 MMLV Q79D CCCCTGGTCCAGCAAGCGAT Btm SDM CAATATGTGGCTTAATCCCC AGGCG 283 MMLV Q79N CGCCTGGGGATTAAGCCACA Top SDM TATTAACCGCTTGCTGGACC AGGGG 284 MMLV Q79N CCCCTGGTCCAGCAAGCGGT Btm SDM TAATATGTGGCTTAATCCCC AGGCG 285 MMLV Q79K CGCCTGGGGATTAAGCCACA Top SDM TATTAAACGCTTGCTGGACC AGGGG 286 MMLV Q79K CCCCTGGTCCAGCAAGCGTT Btm SDM TAATATGTGGCTTAATCCCC AGGCG 287 MMLV L99G CCGTGGAACACCCCCCTTGG Top SDM CCCCGTGAAAAAGCCAGGTA CAAAC 288 MMLV L99G GTTTGTACCTGGCTTTTTCA Btm CGGGGCCAAGGGGGG SDM TGTTCCACGG 289 MMLV L99I CCGTGGAACACCCCCCTTAT Top SDM TCCCGTGAAAAAGCCAGGTA CAAAC 290 MMLV L99I GTTTGTACCTGGCTTTTTCA Btm SDM CGGGAATAAGGGGGGTGTTC CACGG 291 MMLV L99V CCGTGGAACACCCCCCTTGT Top SDM GCCCGTGAAAAAGCCAGGTA CAAAC 292 MMLV L99V GTTTGTACCTGGCTTTTTCA Btm SDM CGGGCACAAGGGGGGTGTTC CACGG 293 MMLV L99P CCGTGGAACACCCCCCTTCC Top SDM GCCCGTGAAAAAGCCAGGTA CAAAC 294 MMLV L99P GTTTGTACCTGGCTTTTTCA Btm SDM CGGGCGGAAGGGGGGTGTTC CACGG 295 MMLV L99M CCGTGGAACACCCCCCTTAT Top SDM GCCCGTGAAAAAGCCAGGTA CAAAC 296 MMLV L99M GTTTGTACCTGGCTTTTTCA Btm SDM CGGGCATAAGGGGGGTGTTC CACGG 297 MMLV L99S CCGTGGAACACCCCCCTTAG Top SDM CCCCGTGAAAAAGCCAGGTA CAAAC 298 MMLV L99S GTTTGTACCTGGCTTTTTCA Btm SDM CGGGGCTAAGGGGGGTGTTC CACGG 299 MMLV L99T CCGTGGAACACCCCCCTTAC Top SDM CCCCGTGAAAAAGCCAGGTA CAAAC 300 MMLV L99T GTTTGTACCTGGCTTTTTCA Btm SDM CGGGGGTAAGGGGGGTGTTC CACGG 301 MMLV L99C CCGTGGAACACCCCCCTTTG Top SDM CCCCGTGAAAAAGCCAGGTA CAAAC 302 MMLV L99C GTTTGTACCTGGCTTTTTCA Btm SDM CGGGGCAAAGGGGGGTGTTC CACGG 303 MMLV L99F CCGTGGAACACCCCCCTTTT Top SDM TCCCGTGAAAAAGCCAGGTA CAAAC 304 MMLV L99F GTTTGTACCTGGCTTTTTCA Btm SDM CGGGAAAAAGGGGGGTGTTC CACGG 305 MMLV L99Y CCGTGGAACACCCCCCTTTA Top SDM TCCCGTGAAAAAGCCAGGTA CAAAC 306 MMLV L99Y GTTTGTACCTGGCTTTTTCA Btm SDM CGGGATAAAGGGGGGTGTTC CACGG 307 MMLV L99H CCGTGGAACACCCCCCTTCA Top SDM TCCCGTGAAAAAGCCAGGTA CAAAC 308 MMLV L99H GTTTGTACCTGGCTTTTTCA Btm CGGGATGAAGGGGGG SDM TGTTCCACGG 309 MMLV L99W CCGTGGAACACCCCCCTTTG Top SDM GCCCGTGAAAAAGCCAGGTA CAAAC 310 MMLV L99W GTTTGTACCTGGCTTTTTCA Btm SDM CGGGCCAAAGGGGGGTGTTC CACGG 311 MMLV L99D CCGTGGAACACCCCCCTTGA Top SDM TCCCGTGAAAAAGCCAGGTA CAAAC 312 MMLV L99D GTTTGTACCTGGCTTTTTCA Btm SDM CGGGATCAAGGGGGGTGTTC CACGG 313 MMLV L99N CCGTGGAACACCCCCCTTAA Top SDM CCCCGTGAAAAAGCCAGGTA CAAAC 314 MMLV L99N GTTTGTACCTGGCTTTTTCA Btm SDM CGGGGTTAAGGGGGGTGTTC CACGG 315 MMLV L99Q CCGTGGAACACCCCCCTTCA Top SDM GCCCGTGAAAAAGCCAGGTA CAAAC 316 MMLV L99Q GTTTGTACCTGGCTTTTTCA Btm SDM CGGGCTGAAGGGGGGTGTTC CACGG 317 MMLV L99K CCGTGGAACACCCCCCTTAA Top SDM ACCCGTGAAAAAGCCAGGTA CAAAC 318 MMLV L99K GTTTGTACCTGGCTTTTTCA Btm SDM CGGGTTTAAGGGGGGTGTTC CACGG 319 MMLV E282G AGAAGGTCAACGTTGGCTGA Top SDM CTGGCGCGCGTAAGGAGACC GTAATG 320 MMLV E282G CATTACGGTCTCCTTACGCG Btm SDM CGCCAGTCAGCCAACGTTGA CCTTCT 321 MMLV E282L AGAAGGTCAACGTTGGCTGA Top SDM CTCTGGCGCGTAAGGAGACC GTAATG 322 MMLV E282L CATTACGGTCTCCTTACGCG Btm SDM CCAGAGTCAGCCAACGTTGA CCTTCT 323 MMLV E282I AGAAGGTCAACGTTGGCTGA Top SDM CTATTGCGCGTAAGGAGACC GTAATG 324 MMLV E282I CATTACGGTCTCCTTACGCG Btm SDM CAATAGTCAGCCAACGTTGA CCTTCT 325 MMLV E282V AGAAGGTCAACGTTGGCTGA Top SDM CTGTGGCGCGTAAGGAGACC GTAATG 326 MMLV E282V CATTACGGTCTCCTTACGCG Btm SDM CCACAGTCAGCCAACGTTGA CCTTCT 327 MMLV E282R AGAAGGTCAACGTTGGCTGA Top SDM CTCCGGCGCGTAAGGAGACC GTAATG 328 MMLV E282P CATTACGGTCTCCTTACGCG Btm CCGGAGTCAGCCAAC SDM GTTGACCTTCT 329 MMLV E282M AGAAGGTCAACGTTGGCTGA Top SDM CTATGGCGCGTAAGGAGACC GTAATG 330 MMLV E282M CATTACGGTCTCCTTACGCG Btm SDM CCATAGTCAGCCAACGTTGA CCTTCT 331 MMLV E282S AGAAGGTCAACGTTGGCTGA Top SDM CTAGCGCGCGTAAGGAGACC GTAATG 332 MMLV E282S CATTACGGTCTCCTTACGCG Btm SDM CGCTAGTCAGCCAACGTTGA CCTTCT 333 MMLV E282T AGAAGGTCAACGTTGGCTGA Top SDM CTACCGCGCGTAAGGAGACC GTAATG 334 MMLV E282T CATTACGGTCTCCTTACGCG Btm SDM CGGTAGTCAGCCAACGTTGA CCTTCT 335 MMLV E282C AGAAGGTCAACGTTGGCTGA Top SDM CTTGCGCGCGTAAGGAGACC GTAATG 336 MMLV E282C CATTACGGTCTCCTTACGCG Btm SDM CGCAAGTCAGCCAACGTTGA CCTTCT 337 MMLV E282F AGAAGGTCAACGTTGGCTGA Top SDM CTTTTGCGCGTAAGGAGACC GTAATG 338 MMLV E282F CATTACGGTCTCCTTACGCG Btm SDM CAAAAGTCAGCCAACGTTGA CCTTCT 339 MMLV E282Y AGAAGGTCAACGTTGGCTGA Top SDM CTTATGCGCGTAAGGAGACC GTAATG 340 MMLV E282Y CATTACGGTCTCCTTACGCG Btm SDM CATAAGTCAGCCAACGTTGA CCTTCT 341 MMLV E282H AGAAGGTCAACGTTGGCTGA Top SDM CTCATGCGCGTAAGGAGACC GTAATG 342 MMLV E282H CATTACGGTCTCCTTACGCG Btm SDM CATGAGTCAGCCAACGTTGA CCTTCT 343 MMLV E282W AGAAGGTCAACGTTGGCTGA Top SDM CTTGGGCGCGTAAGGAGACC GTAATG 344 MMLV E282W CATTACGGTCTCCTTACGCG Btm SDM CCCAAGTCAGCCAACGTTGA CCTTCT 345 MMLV E282N AGAAGGTCAACGTTGGCTGA Top SDM CTAACGCGCGTAAGGAGACC GTAATG 346 MMLV E282N CATTACGGTCTCCTTACGCG Btm SDM CGTTAGTCAGCCAACGTTGA CCTTCT 347 MMLV E282Q AGAAGGTCAACGTTGGCTGA Top SDM CTCAGGCGCGTAAGGAGACC GTAATG 348 MMLV E282Q CATTACGGTCTCCTTACGCG Btm CCTGAGTCAGCCAAC SDM GTTGACCTTCT 349 MMLV E282K AGAAGGTCAACGTTGGCTGA Top SDM CTAAAGCGCGTAAGGAGACC GTAATG 350 MMLV E282K CATTACGGTCTCCTTACGCG Btm SDM CTTTAGTCAGCCAACGTTGA CCTTCT 351 MMLV R298G GCCTACGCCTAAGACGCCAG Top SDM GCCAGTTGCGTGAATTTTTG GGCACAG 352 MMLV R298G CTGTGCCCAAAAATTCACGC Btm SDM AACTGGCCTGGCGTCTTAGG CGTAGGC 353 MMLV R298L GCCTACGCCTAAGACGCCAC Top SDM TGCAGTTGCGTGAATTTTTG GGCACAG 354 MMLV R298L CTGTGCCCAAAAATTCACGC Btm SDM AACTGCAGTGGCGTCTTAGG CGTAGGC 355 MMLV R298I GCCTACGCCTAAGACGCCAA Top SDM TTCAGTTGCGTGAATTTTTG GGCACAG 356 MMLV R298I CTGTGCCCAAAAATTCACGC Btm SDM AACTGAATTGGCGTCTTAGG CGTAGGC 357 MMLV R298V GCCTACGCCTAAGACGCCAG Top SDM TGCAGTTGCGTGAATTTTTG GGCACAG 358 MMLV R298V CTGTGCCCAAAAATTCACGC Btm SDM AACTGCACTGGCGTCTTAGG CGTAGGC 359 MMLV R298P GCCTACGCCTAAGACGCCAC Top SDM CGCAGTTGCGTGAATTTTTG GGCACAG 360 MMLV R298P CTGTGCCCAAAAATTCACGC Btm SDM AACTGCGGTGGCGTCTTAGG CGTAGGC 361 MMLV R298M GCCTACGCCTAAGACGCCAA Top SDM TGCAGTTGCGTGAATTTTTG GGCACAG 362 MMLV R298M CTGTGCCCAAAAATTCACGC Btm SDM AACTGCATTGGCGTCTTAGG CGTAGGC 363 MMLV R298S GCCTACGCCTAAGACGCCAA Top SDM GCCAGTTGCGTGAATTTTTG GGCACAG 364 MMLV R298S CTGTGCCCAAAAATTCACGC Btm SDM AACTGGCTTGGCGTCTTAGG CGTAGGC 365 MMLV R298T GCCTACGCCTAAGACGCCAA Top SDM CCCAGTTGCGTGAATTTTTG GGCACAG 366 MMLV R298T CTGTGCCCAAAAATTCACGC Btm SDM AACTGGGTTGGCGTCTTAGG CGTAGGC 367 MMLV R298C GCCTACGCCTAAGACGCCAT Top SDM GCCAGTTGCGTGAATTTTTG GGCACAG 368 MMLV R298C CTGTGCCCAAAAATTCACGC Btm AACTGGCATGGCGTC SDM TTAGGCGTAGGC 369 MMLV R298F GCCTACGCCTAAGACGCCAT Top SDM TTCAGTTGCGTGAATTTTTG GGCACAG 370 MMLV R298F CTGTGCCCAAAAATTCACGC Btm SDM AACTGAAATGGCGTCTTAGG CGTAGGC 371 MMLV R298Y GCCTACGCCTAAGACGCCAT Top SDM ATCAGTTGCGTGAATTTTTG GGCACAG 372 MMLV R298Y CTGTGCCCAAAAATTCACGC Btm SDM AACTGATATGGCGTCTTAGG CGTAGGC 373 MMLV R298H GCCTACGCCTAAGACGCCAC Top SDM ATCAGTTGCGTGAATTTTTG GGCACAG 374 MMLV R298H CTGTGCCCAAAAATTCACGC Btm SDM AACTGATGTGGCGTCTTAGG CGTAGGC 375 MMLV R298W GCCTACGCCTAAGACGCCAT Top SDM GGCAGTTGCGTGAATTTTTG GGCACAG 376 MMLV R298W CTGTGCCCAAAAATTCACGC Btm SDM AACTGCCATGGCGTCTTAGG CGTAGGC 377 MMLV R298D GCCTACGCCTAAGACGCCAG Top SDM ATCAGTTGCGTGAATTTTTG GGCACAG 378 MMLV R298D CTGTGCCCAAAAATTCACGC Btm SDM AACTGATCTGGCGTCTTAGG CGTAGGC 379 MMLV R298N GCCTACGCCTAAGACGCCAA Top SDM ACCAGTTGCGTGAATTTTTG GGCACAG 380 MMLV R298N CTGTGCCCAAAAATTCACGC Btm SDM AACTGGTTTGGCGTCTTAGG CGTAGGC 381 MMLV R298Q GCCTACGCCTAAGACGCCAC Top SDM AGCAGTTGCGTGAATTTTTG GGCACAG 382 MMLV R298Q CTGTGCCCAAAAATTCACGC Btm SDM AACTGCTGTGGCGTCTTAGG CGTAGGC 383 MMLV I61R/Q68R AGGCAACGTCTACACCTGTC Top SDM TCTCGTAAACAGTACCCCAT GAGTCGTGAGGCCCGCCTGG GG 384 MMLV I61R/Q68R CCCCAGGCGGGCCTCACGAC Btm SDM TCATGGGGTACTGTTTACGA GAGACAGGTGTAGACGTTGC CT 385 MMLV I61K/Q68R AGGCAACGTCTACACCTGTC Top SDM TCTAAAAAACAGTACCCCAT GAGTCGTGAGG 386 MMLV I61K/Q68R CCTCACGACTCATGGGGTAC Btm SDM TGTTTTTTAGAGACAGGTGT AGACGTTGCCT 387 MMLV I61M/Q68R AGGCAACGTCTACACCTGTC Top SDM TCTATGAAACAGTACCCCAT GAGTCGTGAGG 388 MMLV I61M/Q68R CCTCACGACTCATGGGGTAC TGTTTCATAGAGACA Btm SDM GGTGTAGACGTTGCCT 389 MMLV I61M/Q68I AGGCAACGTCTACACCTGTC Top SDM TCTATGAAACAGTACCCCAT GAGTATTGAGGCC 390 MMLV I61M/Q68I GGCCTCAATACTCATGGGGT Btm SDM ACTGTTTCATAGAGACAGGT GTAGACGTTGCCT 393 MMLV 5′ Primer GTCTCTATCAAACAGTACCC CATGGCGCAAGAGGCCCGCC TGGG 394 MMLV 3’ Primer GTCTCTATCAAACAGTACCC CATGCGTCAAGAGGCCCGCC TGGG 395 MMLV G73A CATGAGTCAAGAGGCCCGCG Top SDM AGGGGATTAAGCCACATATT CAGCG 396 MMLV G73R GAGTCAAGAGGCCCGCCTGG Top SDM CGATTAAGCCACATATTCAG CGCTTGC 397 MMLV G73E GAGTCAAGAGGCCCGCCTGC Top SDM GTATTAAGCCACATATTCAG CGCTTGC 398 MMLV P76A GAGTCAAGAGGCCCGCCTGG Top SDM AGATTAAGCCACATATTCAG CGCTTGC 399 MMLV P76R GGCCCGCCTGGGGATTAAGG Top SDM CGCATATTCAGCGCTTGCTG GACC 400 MMLV P76E GGCCCGCCTGGGGATTAAGC Top SDM GTCATATTCAGCGCTTGCTG GACC 401 MMLV H77A GGCCCGCCTGGGGATTAAGG Top SDM AGCATATTCAGCGCTTGCTG GACC 402 MMLV H77R CCGCCTGGGGATTAAGCCAG Top SDM CGATTCAGCGCTTGCTGGAC CAG 403 MMLV H77E CCGCCTGGGGATTAAGCCAC Top SDM GTATTCAGCGCTTGCTGGAC CAG 404 MMLV L82A CCGCCTGGGGATTAAGCCAG Top SDM AGATTCAGCGCTTGCTGGAC CAG 405 MMLV L82R GATTAAGCCACATATTCAGC Top SDM GCTTGGCGGACCAGGGGATC TTGGTCC 406 MMLV L82E GATTAAGCCACATATTCAGC Top SDM GCTTGCGTGACCAGGGGATC TTGGTCC 407 MMLV D83A GATTAAGCCACATATTCAGC Top SDM GCTTGGAGGACCAGGGGATC TTGGTCC 408 MMLV D83R GCCACATATTCAGCGCTTGC Top SDM TGGCGCAGGGGATCTTGGTC CCATG 409 MMLV D83E GCCACATATTCAGCGCTTGC Top SDM TGCGTCAGGGGATCTTGGTC CCATG 410 MMLV I125A GCCACATATTCAGCGCTTGC Top TGGAGCAGGGGATCT SDM TGGTCCCATG 411 MMLV I125R AGGTCAACAAACGCGTAGAA Top SDM GACGCGCATCCGACTGTACC TAATCCTTATAAT 412 MMLV I125E AGGTCAACAAACGCGTAGAA Top SDM GACCGTCATCCGACTGTACC TAATCCTTATAAT 413 MMLV V129A AGGTCAACAAACGCGTAGAA Top SDM GACGAGCATCCGACTGTACC TAATCCTTATAAT 414 MMLV V129R GCGTAGAAGACATCCATCCG Top SDM ACTGCGCCTAATCCTTATAA TCTGTTATCAGGC 415 MMLV V129E GCGTAGAAGACATCCATCCG Top SDM ACTCGTCCTAATCCTTATAA TCTGTTATCAGGC 416 MMLV LI 98A GCGTAGAAGACATCCATCCG Top SDM ACTGAGCCTAATCCTTATAA TCTGTTATCAGGC 417 MMLV LI98R AGGGCTTTAAAAACAGCCCC Top SDM ACAGCGTTCGATGAAGCACT TCACCGTGA 418 MMLV L198E AGGGCTTTAAAAACAGCCCC Top SDM ACACGTTTCGATGAAGCACT TCACCGTGA 419 MMLV E201A AGGGCTTTAAAAACAGCCCC Top SDM ACAGAGTTCGATGAAGCACT TCACCGTGA 420 MMLV E201R TTTAAAAACAGCCCCACATT Top SDM GTTCGATGCGGCACTTCACC GTGACTTAGCAG 421 MMLV E201D TTTAAAAACAGCCCCACATT Top SDM GTTCGATCGTGCACTTCACC GTGACTTAGCAG 422 MMLV R205A TTTAAAAACAGCCCCACATT Top SDM GTTCGATGATGCACTTCACC GTGACTTAGCAG 423 MMLV R205K CACATTGTTCGATGAAGCAC Top SDM TTCACGCGGACTTAGCAGAC TTCCGTATCCA 424 MMLV R205E CACATTGTTCGATGAAGCAC Top SDM TTCACAAAGACTTAGCAGAC TTCCGTATCCA 425 MMLV D209A GATGAAGCACTTCACCGTGA Top SDM CTTAGAGGACTTCCGTATCC AACACCCAG 426 MMLV D209R AAGCACTTCACCGTGACTTA Top SDM GCAGCGTTCCGTATCCAACA CCCAGACTT 427 MMLV D209E AAGCACTTCACCGTGACTTA Top SDM GCACGTTTCCGTATCCAACA CCCAGACTT 428 MMLV F210A AAGCACTTCACCGTGACTTA Top SDM GCAGAGTTCCGTATCCAACA CCCAGACTT 429 MMLV F2I0R CACTTCACCGTGACTTAGCA Top SDM GACGCGCGTATCCAACACCC AGACTTAATTC 430 MMLV F210E CACTTCACCGTGACTTAGCA Top GACCGTCGTATCCAA SDM CACCCAGACTTAATTC 431 MMLV R211A CACTTCACCGTGACTTAGCA Top SDM GACGAGCGTATCCAACACCC AGACTTAATTC 432 MMLV R211K TTCACCGTGACTTAGCAGAC Top SDM TTCGCGATCCAACACCCAGA CTTAATTCTGTTA 433 MMLV R21 IE TTCACCGTGACTTAGCAGAC Top SDM TTCAAAATCCAACACCCAGA CTTAATTCTGTTA 434 MMLV 1212A TTCACCGTGACTTAGCAGAC Top SDM TTCGAGATCCAACACCCAGA CTTAATTCTGTTA 435 MMLV I212R CCGTGACTTAGCAGACTTCC Top SDM GTGCGCAACACCCAGACTTA ATTCTGTTACAG 436 MMLV I212E CCGTGACTTAGCAGACTTCC Top SDM GTCGTCAACACCCAGACTTA ATTCTGTTACAG 437 MMLV Q213A CCGTGACTTAGCAGACTTCC Top SDM GTGAGCAACACCCAGACTTA ATTCTGTTACAG 438 MMLV Q213R GTGACTTAGCAGACTTCCGT Top SDM ATCGCGCACCCAGACTTAAT TCTGTTACAGTAT 439 MMLV Q213E GTGACTTAGCAGACTTCCGT Top SDM ATCCGTCACCCAGACTTAAT TCTGTTACAGTAT 440 MMLV K348A GTGACTTAGCAGACTTCCGT Top SDM ATCGAGCACCCAGACTTAAT TCTGTTACAGTAT 441 MMLV K348R AGCAAAAGGCGTATCAGGAG Top SDM ATCGCGCAAGCTTTGTTGAC CGCACCC 442 MMLV K348E AGCAAAAGGCGTATCAGGAG Top SDM ATCCGTCAAGCTTTGTTGAC CGCACCC 443 MMLV L352A AGCAAAAGGCGTATCAGGAG Top SDM ATCGAGCAAGCTTTGTTGAC CGCACCC 444 MMLV L352R CGTATCAGGAGATCAAACAA Top SDM GCTTTGGCGACCGCACCCGC GTTGGG 445 MMLV L352E CGTATCAGGAGATCAAACAA Top SDM GCTTTGCGTACCGCACCCGC GTTGGG 446 MMLV K285A CGTATCAGGAGATCAAACAA Top SDM GCTTTGGAGACCGCACCCGC GTTGGG 447 MMLV K285R GTTGGCTGACTGAAGCGCGT Top SDM GCGGAGACCGTAATGGGGCA GC 448 MMLV K285E GTTGGCTGACTGAAGCGCGT Top SDM CGTGAGACCGTAATGGGGCA GC 449 MMLV Q299A GTTGGCTGACTGAAGCGCGT Top SDM GAGGAGACCGTAATGGGGCA GC 450 MMLV Q299R TACGCCTAAGACGCCACGCG CGTTGCGTGAATTTT Top SDM TGGGCACAGC 451 MMLV Q299E TACGCCTAAGACGCCACGCC Top SDM GTTTGCGTGAATTTTTGGGC ACAGC 452 MMLV G308A TACGCCTAAGACGCCACGCG Top SDM AGTTGCGTGAATTTTTGGGC ACAGC 453 MMLV G308R GCGTGAATTTTTGGGCACAG Top SDM CGGCGTTCTGTCGTTTATGG ATTCCTGGG 454 MMLV G308E GCGTGAATTTTTGGGCACAG Top SDM CGCGTTTCTGTCGTTTATGG ATTCCTGGG 455 MMLV R311A GCGTGAATTTTTGGGCACAG Top SDM CGGAGTTCTGTCGTTTATGG ATTCCTGGG 456 MMLV R311K GGGCACAGCGGGATTCTGTG Top SDM CGTTATGGATTCCTGGGTTC GCTGA 457 MMLV R311E GGGCACAGCGGGATTCTGTA Top SDM AATTATGGATTCCTGGGTTC GCTGA 458 MMLV Y271A GGGCACAGCGGGATTCTGTG Top SDM AGTTATGGATTCCTGGGTTC GCTGA 459 MMLV Y271R GTCAAAAACAGGTAAAGTAC Top SDM CTTGGGGCGTTGCTGAAAGA AGGTCAACGTTGG 460 MMLV Y271E GTCAAAAACAGGTAAAGTAC Top SDM CTTGGGCGTTTGCTGAAAGA AGGTCAACGTTGG 461 MMLV L280A GTCAAAAACAGGTAAAGTAC Top SDM CTTGGGGAGTTGCTGAAAGA AGGTCAACGTTGG 462 MMLV L280R TGCTGAAAGAAGGTCAACGT Top SDM TGGGCGACTGAAGCGCGTAA GGAGACC 463 MMLV L280E TGCTGAAAGAAGGTCAACGT Top SDM TGGCGTACTGAAGCGCGTAA GGAGACC 464 MMLV L357A TGCTGAAAGAAGGTCAACGT Top SDM TGGGAGACTGAAGCGCGTAA GGAGACC 465 MMLV L357R TTTGTTGACCGCACCCGCGG Top SDM CGGGTCTTCCGGATTTAACC AAGCC 466 MMLV L357E TTTGTTGACCGCACCCGCGC Top SDM GTGGTCTTCCGGATTTAACC AAGCC 467 MMLV T328A TTTGTTGACCGCACCCGCGG Top SDM AGGGTCTTCCGGATTTAACC AAGCC 468 MMLV T328R CTGCACCCCTGTACCCCTTA Top SDM GCGAAAACAGGGACGCTTTT CAACTGG 469 MMLV T328E CTGCACCCCTGTACCCCTTA Top SDM CGTAAAACAGGGACGCTTTT CAACTGG 470 MMLV G331A CTGCACCCCTGTACCCCTTA GAGAAAACAGGGACG Top SDM CTTTTCAACTGG 471 MMLV G331R CCCCTGTACCCCTTAACAAA Top SDM AACAGCGACGCTTTTCAACT GGGGGCC 472 MMLV G331E CCCCTGTACCCCTTAACAAA Top SDM AACACGTACGCTTTTCAACT GGGGGCC 473 MMLV T332A CCCCTGTACCCCTTAACT'L Top SDM AAAACAGAGACGCTTTTCAA CTGGGGGCC 474 MMLV T332R CTGTACCCCTTAACAAAAAC Top SDM AGGGGCGCTTTTCAACTGGG GGCCAGAC 475 MMLV T332E CTGTACCCCTTAACAAAAAC Top SDM AGGGCGTCTTTTCAACTGGG GGCCAGAC 476 MMLV N335A CTGTACCCCTTAACAAAAAC Top SDM AGGGGAGCTTTTCAACTGGG GGCCAGAC All MMLV N335R CCTTAACAAAAACAGGGACG Top SDM CTTTTCGCGTGGGGGCCAGA CCAGCAAA 478 MMLV N335E CCTTAACAAAAACAGGGACG Top SDM CTTTTCCGTTGGGGGCCAGA CCAGCAAA 479 MMLV E367A CTTCCGGATTTAACCAAGCC Top SDM CTTTGCGCTGTTCGTTGATG AAAAACAGGGATAT 480 MMLV E367R CTTCCGGATTTAACCAAGCC Top SDM CTTTCGTCTGTTCGTTGATG AAAAACAGGGATAT 481 MMLV E367D CTTCCGGATTTAACCAAGCC Top SDM CTTTGATCTGTTCGTTGATG AAAAACAGGGATAT 482 MMLV F369A GATTTAACCAAGCCCTTTGA Top SDM GCTGGCGGTTGATGAAAAAC AGGGATATGCAAAAG 483 MMLV F369R GATTTAACCAAGCCCTTTGA Top SDM GCTGCGTGTTGATGAAAAAC AGGGATATGCAAAAG 484 MMLV F369E GATTTAACCAAGCCCTTTGA Top SDM GCTGGAGGTTGATGAAAAAC AGGGATATGCAAAAG 485 MMLV R389A CCCAAAAGTTAGGCCCGTGG Top SDM GCGCGCCCTGTTGCTTACTT GAGTAA 486 MMLV R389K CCCAAAAGTTAGGCCCGTGG Top SDM AAACGCCCTGTTGCTTACTT GAGTAA 487 MMLV R389E CCCAAAAGTTAGGCCCGTGG Top SDM GAGCGCCCTGTTGCTTACTT GAGTAA 488 MMLV V433A AGTTGACGATGGGTCAACCC Top SDM TTAGCGATCTTGGCTCCACA TGCTGTAGA 489 MMLV V433R AGTTGACGATGGGTCAACCC Top SDM TTACGTATCTTGGCTCCACA TGCTGTAGA 490 MMLV V433E AGTTGACGATGGGTCAACCC Top TTAGAGATCTTGGCT SDM CCACATGCTGTAGA 491 MMLV V476A GGATCGTGTACAATTTGGAC Top SDM CAGTTGCGGCTTTGAATCCA GCTACTTTGCTTC 492 MMLV V476R GGATCGTGTACAATTTGGAC Top SDM CAGTTCGTGCTTTGAATCCA GCTACTTTGCTTC 493 MMLV V476E GGATCGTGTACAATTTGGAC Top SDM CAGTTGAGGCTTTGAATCCA GCTACTTTGCTTC 494 MMLV 1593A CGTTATGCTTTTGCAACAGC Top SDM GCATGCGCATGGCGAAATTT ACCGCCGC 495 MMLV 1593R CGTTATGCTTTTGCAACAGC Top SDM GCATCGTCATGGCGAAATTT ACCGCCGC 496 MMLV I593E CGTTATGCTTTTGCAACAGC Top SDM GCATGAGCATGGCGAAATTT ACCGCCGC 497 MMLV E596A GCAACAGCGCATATCCATGG Top SDM CGCGATTTACCGCCGCCGTG GTC 498 MMLV E596R GCAACAGCGCATATCCATGG Top SDM CCGTATTTACCGCCGCCGTG GTC 499 MMLV E596D GCAACAGCGCATATCCATGG Top SDM CGATATTTACCGCCGCCGTG GTC 500 MMLV 1597A CAACAGCGCATATCCATGGC Top SDM GAAGCGTACCGCCGCCGTGG TCTG 501 MMLV 1597R CAACAGCGCATATCCATGGC Top SDM GAACGTTACCGCCGCCGTGG TCTG 502 MMLV I597E CAACAGCGCATATCCATGGC Top SDM GAAGAGTACCGCCGCCGTGG TCTG 503 MMLV R650A AGCGGAGGCTCGTGGAAACG Top SDM CGATGGCGGACCAAGCTGCC C 504 MMLV R650K AGCGGAGGCTCGTGGAAACA Top SDM AAATGGCGGACCAAGCTGCC C 505 MMLV R650E AGCGGAGGCTCGTGGAAACG Top SDM AGATGGCGGACCAAGCTGCC C 506 MMLV Q654A GTGGAAACCGTATGGCGGAC Top SDM GCGGCTGCCCGTAAGGCGGC 507 MMLV Q654R GTGGAAACCGTATGGCGGAC Top SDM CGTGCTGCCCGTAAGGCGGC 508 MMLV Q654E GTGGAAACCGTATGGCGGAC Top SDM GAGGCTGCCCGTAAGGCGGC 509 MMLV R657A TATGGCGGACCAAGCTGCCG Top SDM CGAAGGCGGCGATCACAGAG AC 510 MMLV R657K TATGGCGGACCAAGCTGCCA AAAAGGCGGCGATCA Top SDM CAGAGAC 511 MMLV R657E TATGGCGGACCAAGCTGCCG Top SDM AGAAGGCGGCGATCACAGAG AC 512 MMLV G73A GCAAGCGCTGAATATGTGGC Btm SDM TTAATCGCCAGGCGGGCCTC TTGACTC 513 MMLV G73R GCAAGCGCTGAATATGTGGC Btm SDM TTAATACGCAGGCGGGCCTC TTGACTC 514 MMLV G73E GCAAGCGCTGAATATGTGGC Btm SDM TTAATCTCCAGGCGGGCCTC TTGACTC 515 MMLV P76A GGTCCAGCAAGCGCTGAATA Btm SDM TGCGCCTTAATCCCCAGGCG GGCC 516 MMLV P76R GGTCCAGCAAGCGCTGAATA Btm SDM TGACGCTTAATCCCCAGGCG GGCC 517 MMLV P76E GGTCCAGCAAGCGCTGAATA Btm SDM TGCTCCTTAATCCCCAGGCG GGCC 518 MMLV H77A CTGGTCCAGCAAGCGCTGAA Btm SDM TCGCTGGCTTAATCCCCAGG CGG 519 MMLV H77R CTGGTCCAGCAAGCGCTGAA Btm SDM TACGTGGCTTAATCCCCAGG CGG 520 MMLV H77E CTGGTCCAGCAAGCGCTGAA Btm SDM TCTCTGGCTTAATCCCCAGG CGG 521 MMLV L82A GGACCAAGATCCCCTGGTCC Btm SDM GCCAAGCGCTGAATATGTGG CTTAATC 522 MMLV L82R GGACCAAGATCCCCTGGTCA Btm SDM CGCAAGCGCTGAATATGTGG CTTAATC 523 MMLV L82E GGACCAAGATCCCCTGGTCC Btm SDM TCCAAGCGCTGAATATGTGG CTTAATC 524 MMLV D83A CATGGGACCAAGATCCCCTG Btm SDM CGCCAGCAAGCGCTGAATAT GTGGC 525 MMLV D83R CATGGGACCAAGATCCCCTG Btm SDM ACGCAGCAAGCGCTGAATAT GTGGC 526 MMLV D83E CATGGGACCAAGATCCCCTG Btm SDM CTCCAGCAAGCGCTGAATAT GTGGC 527 MMLV I125A ATTATAAGGATTAGGTACAG Btm SDM TCGGATGCGCGTCTTCTACG CGTTTGTTGACCT 528 MMLV I125R ATTATAAGGATTAGGTACAG Btm SDM TCGGATGACGGTCTTCTACG CGTTTGTTGACCT 529 MMLV I125E ATTATAAGGATTAGGTACAG Btm SDM TCGGATGCTCGTCTTCTACG CGTTTGTTGACCT 530 MMLV VI29A GCCTGATAACAGATTATAAG GATTAGGCGCAGTCG Btm SDM GATGGATGTCTTCTACGC 531 MMLV V129R GCCTGATAACAGATTATAAG Btm SDM GATTAGGACGAGTCGGATGG ATGTCTTCTACGC 532 MMLV V129E GCCTGATAACAGATTATAAG Btm SDM GATTAGGCTCAGTCGGATGG ATGTCTTCTACGC 533 MMLV L198A TCACGGTGAAGTGCTTCATC Btm SDM GAACGCTGTGGGGCTGTTTT TAAAGCCCT 534 MMLV L198R TCACGGTGAAGTGCTTCATC Btm SDM GAAACGTGTGGGGCTGTTTT TAAAGCCCT 535 MMLV L198E TCACGGTGAAGTGCTTCATC Btm SDM GAACTCTGTGGGGCTGTTTT TAAAGCCCT 536 MMLV E201A CTGCTAAGTCACGGTGAAGT Btm SDM GCCGCATCGAACAATGTGGG GCTGTTTTTAAA 537 MMLV E201R CTGCTAAGTCACGGTGAAGT Btm SDM GCACGATCGAACAATGTGGG GCTGTTTTTAAA 538 MMLV E201D CTGCTAAGTCACGGTGAAGT Btm SDM GCATCATCGAACAATGTGGG GCTGTTTTTAAA 539 MMLV R205A TGGATACGGAAGTCTGCTAA Btm SDM GTCCGCGTGAAGTGCTTCAT CGAACAATGTG 540 MMLV R205K TGGATACGGAAGTCTGCTAA Btm SDM GTCTTTGTGAAGTGCTTCAT CGAACAATGTG 541 MMLV R205E TGGATACGGAAGTCTGCTAA Btm SDM GTCCTCGTGAAGTGCTTCAT CGAACAATGTG 542 MMLV D209A AAGTCTGGGTGTTGGATACG Btm SDM GAACGCTGCTAAGTCACGGT GAAGTGCTT 543 MMLV D209R AAGTCTGGGTGTTGGATACG Btm SDM GAAACGTGCTAAGTCACGGT GAAGTGCTT 544 MMLV D209E AAGTCTGGGTGTTGGATACG Btm SDM GAACTCTGCTAAGTCACGGT GAAGTGCTT 545 MMLV F210A GAATTAAGTCTGGGTGTTGG Btm SDM ATACGCGCGTCTGCTAAGTC ACGGTGAAGTG 546 MMLV F210R GAATTAAGTCTGGGTGTTGG Btm SDM ATACGACGGTCTGCTAAGTC ACGGTGAAGTG 547 MMLV F210E GAATTAAGTCTGGGTGTTGG Btm SDM ATACGCTCGTCTGCTAAGTC ACGGTGAAGTG 548 MMLV R211A TAACAGAATTAAGTCTGGGT Btm SDM GTTGGATCGCGAAGTCTGCT AAGTCACGGTGAA 549 MMLV R211K TAACAGAATTAAGTCTGGGT Btm SDM GTTGGATTTTGAAGTCTGCT AAGTCACGGTGAA 550 MMLV R211E TAACAGAATTAAGTCTGGGT Btm SDM GTTGGATCTCGAAGT CTGCTAAGTCACGGTGAA 551 MMLV I2I2A CTGTAACAGAATTAAGTCTG Btm SDM GGTGTTGCGCACGGAAGTCT GCTAAGTCACGG 552 MMLV 1212R CTGTAACAGAATTAAGTCTG Btm SDM GGTGTTGACGACGGAAGTCT GCTAAGTCACGG 553 MMLV 1212E CTGTAACAGAATTAAGTCTG Btm SDM GGTGTTGCTCACGGAAGTCT GCTAAGTCACGG 554 MMLV Q213A ATACTGTAACAGAATTAAGT Btm SDM CTGGGTGCGCGATACGGAAG TCTGCTAAGTCAC 555 MMLV Q213R ATACTGTAACAGAATTAAGT Btm SDM CTGGGTGACGGATACGGAAG TCTGCTAAGTCAC 556 MMLV Q213E ATACTGTAACAGAATTAAGT Btm SDM CTGGGTGCTCGATACGGAAG TCTGCTAAGTCAC 557 MMLV K348A GGGTGCGGTCAACAAAGCTT Btm SDM GCGCGATCTCCTGATACGCC TTTTGCT 558 MMLV K348R GGGTGCGGTCAACAAAGCTT Btm SDM GACGGATCTCCTGATACGCC TTTTGCT 559 MMLV K348E GGGTGCGGTCAACAAAGCTT Btm SDM GCTCGATCTCCTGATACGCC TTTTGCT 560 MMLV L352A CCCAACGCGGGTGCGGTCGC Btm SDM CAAAGCTTGTTTGATCTCCT GATACG 561 MMLV L352R CCCAACGCGGGTGCGGTACG Btm SDM CAAAGCTTGTTTGATCTCCT GATACG 562 MMLV L352E CCCAACGCGGGTGCGGTCTC Btm SDM CAAAGCTTGTTTGATCTCCT GATACG 563 MMLV K285A GCTGCCCCATTACGGTCTCC Btm SDM GCACGCGCTTCAGTCAGCCA AC 564 MMLV K285R GCTGCCCCATTACGGTCTCA Btm SDM CGACGCGCTTCAGTCAGCCA AC 565 MMLV K285E GCTGCCCCATTACGGTCTCC Btm SDM TCACGCGCTTCAGTCAGCCA AC 566 MMLV Q299A GCTGTGCCCAAAAATTCACG Btm SDM CAACGCGCGTGGCGTCTTAG GCGTA 567 MMLV Q299R GCTGTGCCCAAAAATTCACG Btm SDM CAAACGGCGTGGCGTCTTAG GCGTA 568 MMLV Q299E GCTGTGCCCAAAAATTCACG Btm SDM CAACTCGCGTGGCGTCTTAG GCGTA 569 MMLV G308A CCCAGGAATCCATAAACGAC Btm SDM AGAACGCCGCTGTGCCCAAA AATTCACGC 570 MMLV G308R CCCAGGAATCCATAAACGAC AGAAACGCGCTGTGC Btm SDM CCAAAAATTCACGC 571 MMLV G308E CCCAGGAATCCATAAACGAC Btm SDM AGAACTCCGCTGTGCCCAAA AATTCACGC 572 MMLV R311A TCAGCGAACCCAGGAATCCA Btm SDM TAACGCACAGAATCCCGCTG TGCCC 573 MMLV R311K TCAGCGAACCCAGGAATCCA Btm SDM TAATTTACAGAATCCCGCTG TGCCC 574 MMLV R311E TCAGCGAACCCAGGAATCCA Btm SDM TAACTCACAGAATCCCGCTG TGCCC 575 MMLV Y271A CCAACGTTGACCTTCTTTCA Btm SDM GCAACGCCCCAAGGTACTTT ACCTGTTTTTGAC 576 MMLV Y271R CCAACGTTGACCTTCTTTCA Btm SDM GCAAACGCCCAAGGTACTTT ACCTGTTTTTGAC 577 MMLV Y271E CCAACGTTGACCTTCTTTCA Btm SDM GCAACTCCCCAAGGTACTTT ACCTGTTTTTGAC 578 MMLV L280A GGTCTCCTTACGCGCTTCAG Btm SDM TCGCCCAACGTTGACCTTCT TTCAGCA 579 MMLV L280R GGTCTCCTTACGCGCTTCAG Btm SDM TACGCCAACGTTGACCTTCT TTCAGCA 580 MMLV L280E GGTCTCCTTACGCGCTTCAG Btm SDM TCTCCCAACGTTGACCTTCT TTCAGCA 581 MMLV L357A GGCTTGGTTAAATCCGGAAG Btm SDM ACCCGCCGCGGGTGCGGTCA ACAAA 582 MMLV L357R GGCTTGGTTAAATCCGGAAG Btm SDM ACCACGCGCGGGTGCGGTCA ACAAA 583 MMLV L357E GGCTTGGTTAAATCCGGAAG Btm SDM ACCCTCCGCGGGTGCGGTCA ACAAA 584 MMLV T328A CCAGTTGAAAAGCGTCCCTG Btm SDM TTTTCGCTAAGGGGTACAGG GGTGCAG 585 MMLV T328R CCAGTTGAAAAGCGTCCCTG Btm SDM TTTTACGTAAGGGGTACAGG GGTGCAG 586 MMLV T328E CCAGTTGAAAAGCGTCCCTG Btm SDM TTTTCTCTAAGGGGTACAGG GGTGCAG 587 MMLV G331A GGCCCCCAGTTGAAAAGCGT Btm SDM CGCTGTTTTTGTTAAGGGGT ACAGGGG 588 MMLV G331R GGCCCCCAGTTGAAAAGCGT Btm SDM ACGTGTTTTTGTTAAGGGGT ACAGGGG 589 MMLV G331E GGCCCCCAGTTGAAAAGCGT Btm SDM CTCTGTTTTTGTTAAGGGGT ACAGGGG 590 MMLV T332A GTCTGGCCCCCAGTTGAAAA GCGCCCCTGTTTTTG Btm SDM TTAAGGGGTACAG 591 MMLV T332R GTCTGGCCCCCAGTTGAAAA Btm SDM GACGCCCTGTTTTTGTTAAG GGGTACAG 592 MMLV T332E GTCTGGCCCCCAGTTGAAAA Btm SDM GCTCCCCTGTTTTTGTTAAG GGGTACAG 593 MMLV N335A TTTGCTGGTCTGGCCCCCAC Btm SDM GCGAAAAGCGTCCCTGTTTT TGTTAAGG 594 MMLV N335R TTTGCTGGTCTGGCCCCCAA Btm SDM CGGAAAAGCGTCCCTGTTTT TGTTAAGG 595 MMLV N335E TTTGCTGGTCTGGCCCCCAC Btm SDM TCGAAAAGCGTCCCTGTTTT TGTTAAGG 596 MMLV E367A ATATCCCTGTTTTTCATCAA Btm SDM CGAACAGCGCAAAGGGCTTG GTTAAATCCGGAAG 597 MMLV E367R ATATCCCTGTTTTTCATCAA Btm SDM CGAACAGACGAAAGGGCTTG GTTAAATCCGGAAG 598 MMLV E367D ATATCCCTGTTTTTCATCAA Btm SDM CGAACAGATCAAAGGGCTTG GTTAAATCCGGAAG 599 MMLV F369A CTTTTGCATATCCCTGTTTT Btm SDM TCATCAACCGCCAGCTCAAA GGGCTTGGTTAAATC 600 MMLV F369R CTTTTGCATATCCCTGTTTT Btm SDM TCATCAACACGCAGCTCAAA GGGCTTGGTTAAATC 601 MMLV F369E CTTTTGCATATCCCTGTTTT Btm SDM TCATCAACCTCCAGCTCAAA GGGCTTGGTTAAATC 602 MMLV R389A TTACTCAAGTAAGCAACAGG Btm SDM GCGCGCCCACGGGCCTAACT TTTGGG 603 MMLV R389K TTACTCAAGTAAGCAACAGG Btm SDM GCGTTTCCACGGGCCTAACT TTTGGG 604 MMLV R389E TTACTCAAGTAAGCAACAGG Btm SDM GCGCTCCCACGGGCCTAACT TTTGGG 605 MMLV V433A TCTACAGCATGTGGAGCCAA Btm SDM GATCGCTAAGGGTTGACCCA TCGTCAACT 606 MMLV V433R TCTACAGCATGTGGAGCCAA Btm SDM GATACGTAAGGGTTGACCCA TCGTCAACT 607 MMLV V433E TCTACAGCATGTGGAGCCAA Btm SDM GATCTCTAAGGGTTGACCCA TCGTCAACT 608 MMLV V476A GAAGCAAAGTAGCTGGATTC Btm SDM AAAGCCGCAACTGGTCCAAA TTGTACACGATCC 609 MMLV V476R GAAGCAAAGTAGCTGGATTC Btm SDM AAAGCACGAACTGGTCCAAA TTGTACACGATCC 610 MMLV V476E GAAGCAAAGTAGCTGGATTC Btm SDM AAAGCCTCAACTGGT CCAAATTGTACACGATCC 611 MMLV 1593A GCGGCGGTAAATTTCGCCAT Btm SDM GCGCATGCGCTGTTGCAAAA GCATAACG 612 MMLV I593R GCGGCGGTAAATTTCGCCAT Btm SDM GACGATGCGCTGTTGCAAAA GCATAACG 613 MMLV I593E GCGGCGGTAAATTTCGCCAT Btm SDM GCTCATGCGCTGTTGCAAAA GCATAACG 614 MMLV E596A GACCACGGCGGCGGTAAATC Btm SDM GCGCCATGGATATGCGCTGT TGC 615 MMLV E596R GACCACGGCGGCGGTAAATA Btm SDM CGGCCATGGATATGCGCTGT TGC 616 MMLV E596D GACCACGGCGGCGGTAAATA Btm SDM TCGCCATGGATATGCGCTGT TGC 617 MMLV 1597A CAGACCACGGCGGCGGTACG Btm SDM CTTCGCCATGGATATGCGCT GTTG 618 MMLV I597R CAGACCACGGCGGCGGTAAC Btm SDM GTTCGCCATGGATATGCGCT GTTG 619 MMLV I597E CAGACCACGGCGGCGGTACT Btm SDM CTTCGCCATGGATATGCGCT GTTG 620 MMLV R650A GGGCAGCTTGGTCCGCCATC Btm SDM GCGTTTCCACGAGCCTCCGC T 621 MMLV R650K GGGCAGCTTGGTCCGCCATT Btm SDM TTGTTTCCACGAGCCTCCGC T 622 MMLV R650E GGGCAGCTTGGTCCGCCATC Btm SDM TCGTTTCCACGAGCCTCCGC T 623 MMLV Q654A GCCGCCTTACGGGCAGCCGC Btm SDM GTCCGCCATACGGTTTCCAC 624 MMLV Q654R GCCGCCTTACGGGCAGCACG Btm SDM GTCCGCCATACGGTTTCCAC 625 MMLV Q654E GCCGCCTTACGGGCAGCCTC Btm SDM GTCCGCCATACGGTTTCCAC 626 MMLV R657A GTCTCTGTGATCGCCGCCTT Btm SDM CGCGGCAGCTTGGTCCGCCA TA 627 MMLV R657K GTCTCTGTGATCGCCGCCTT Btm SDM TTTGGCAGCTTGGTCCGCCA TA 628 MMLV R657E GTCTCTGTGATCGCCGCCTT Btm SDM CTCGGCAGCTTGGTCCGCCA TA 629 MMLV L280R ATTTGCTGAAAGAAGGTCAA Top SDM V2 CGTTGGCGTACTGATGCGCG TAAGGAGACC 630 MMLV L280R GGTCTCCTTACGCGCATCAG Btm SDM V2 TACGCCAACGTTGACCTTCT TTCAGCAAAT 631 MMLV L82R GGGATTAAGCCACATATTCG Top SDM V2 TCGCTTGCGTGACCAGGGGA TCTTGGTCCC 632 MMLV L82R GGGACCAAGATCCCCTGGTC Btm SDM V2 ACGCAAGCGACGAATATGTG GCTTAATCCC

Example 2: Preparation of Reverse Transcriptase Mutants for Screening Increased Activity and Thermostability

a. Overexpression of MMLV RTase and Mutant Variants

A test induction was used to determine optimum growing conditions. A colony, with the appropriate strain, was used to inoculate Terrific Broth (TB) media (50 mL) with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was reached. The 50 mL culture was divided in half to accommodate two induction temperatures. IPTG (1M; 12.5 μL) was used to induce protein expression, followed by growth at two induction temperatures for 21 hours. Aliquots (normalized to an OD of 1.25) were taken at 3 and 21 hours, cells were harvested at 13,000×g for one minute, and harvested cells were stored at −20° C. Cells were resuspended in 1× SDS-PAGE running buffer (270 μL) and 5× SDS-PAGE loading dye (70 μL). Samples were boiled for 5 minutes, sonicated, and loaded (15 μL) onto a 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio Rad, Cat #4568094). SDS-PAGE images are shown in FIG. 2.

b. Expression and Purification of MMLV RTase and Mutant Variants

A colony with the appropriate strain was used to inoculate TB media (1 mL, in a 96-well deep well plate) with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was achieved followed by cooling of the plate on ice for 5 minutes. Protein expression was induced by the addition of 100 mM IPTG (5 μL), followed by growth at 18° C. for 21 hours. Cells were harvested by spinning samples at 4,700×g for 10 minutes.

Cell pellets were re-suspended in a lysis buffer (50 mM NaPO₄, pH 7.8, 5% glycerol, 300 mM NaCl, and 10 mM imidazole) and lysed by the addition of 1× BugBuster® (Millipore Sigma, Cat #70921) and incubation on an end-over-end mixer for 15 minutes at room temperature. Cell debris was removed by centrifuging the lysate at 16,000×g for 20 minutes at 4° C.

Cleared lysates were applied to a HisPur™ Ni-NTA spin plate (ThermoFisher, Cat #88230). Resin was equilibrated with Screening His-Bind buffer (50 mM NaPO₄, pH 7.8, 5% glycerol, 300 mM NaCl, and 10 mM imidazole) and samples loaded. Samples were washed three times with Screening His-Wash buffer (50 mM NaPO₄, pH 7.8, 5% glycerol, 300 mM NaCl, and 25 mM imidazole) and eluted using Screening His-Elution buffer (50 mM NaPO₄, pH 7.8, 5% glycerol, 300 mM NaCl, and 250 mM imidazole). Purified proteins were normalized to a set concentration (100 nM) for testing purposes.

Example 3: Evaluation of Reverse Transcriptase Mutants

a. Evaluation of Ability of RTase Mutants to Synthesize DNA

The ability of mutant RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) was compared to an MMLV RTase base construct (RNase H minus construct). Mutant MMLV RTases were tested in two formats: (1) standard two-step cDNA synthesis with gene specific primers, followed by qPCR, and (2) one-step addition of the RTase in Integrated DNA Technologies PrimeTime® Gene Expression Master Mix (GEM).

b. Standard Two-Step Procedure

RTases (2 μL, 100 nM) were added to a reaction mixture containing RNA (50 ng), dNTPs (100 μM), gene specific primer set (500 nM; see Table 2), first strand synthesis buffer (1×, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT), and SuperaseIN (0.17 U/μL) in a 50 μL volume. The reaction was allowed to proceed at 50° C. for 15 minutes, followed by incubation at 80° C. for 10 minutes.

cDNA synthesized by RTase mutants was quantified by qPCR amplification using an assay that identified the SFRS9 gene in human cells. The assay master mix composition included GEM (1×), ROX (50 nM), SFRS9 primer set (500 nM; see Table 2), and SFRS9 probe (250 nM; see Table 2). Assay master mix and synthesized cDNA were mixed at a 4:1 ratio for a final volume of 20 μL. The reaction was run on qPCR (QuantStudio) for 40 cycles under the following cycle conditions: 95° C. hold for 3 minutes, 95° C. for 15 seconds, and 60° C. for one minute.

TABLE 2 Sequences of primers and probes used for qPCR assays. SEQ Primer Primer Sequence ID NO: Name (5′-3′) 633 Hs SFRS9 GTCGAGTATCTCAGAAAAGAAGACA Forward Primer 634 Hs SFRS9 CTCGGATGTAGGAAGTTTCACC Reverse Primer 635 Hs SFRS9 /5SUN/ATGCCCTGC/ Probe- ZEN/GTAAACTGGATGACA SUN /3IABkFQ/ c. One-Step Procedure in GEM

RTases (1 μL, 100 nM) were added to a reaction mixture containing RNA (10 ng), GEM (1×), ROX (50 nM), SFRS9 primer set (500 nM; see Table 2), and SFRS9 probe (250 nM; see Table 2) in a final volume of 20 μL. The reaction was run on a qPCR machine (QuantStudio) for 40 cycles using the following cycle conditions: 60° C. hold for 15 minutes, 95° C. hold for 3 minutes, 95° C. for 15 seconds, and 60° C. for one minute.

d. MMLV RTase Base Construct and Single Mutant Variants

As described in Example 1, MMLV RTase single mutant variants were prepared by introducing selected mutations into the MMLV RTase base construct by site-directed mutagenesis, using standard PCR conditions and primers. The sequences of the MMLV RTase base construct and single mutant variants are shown in Table 3. One of skill in the art will understand that the MMLV RTase amino acid sequences set forth in SEQ ID NO: 637 and SEQ ID NO: 717 (the latter of which is described in Example 6 below) are truncated forms of the full-length amino acid sequence of wild-type, or naturally occurring, MMLV RTase. In addition, a person having ordinary skill in the art will understand that a methionine residue is required to recombinantly produce the MMLV RTase base construct and mutants of the disclosure, and as such, that the MMLV RTase sequences disclosed herein (see, e.g., Table 3 below, Table 8 in Example 4, Tables 9 and 12 in Example 5, Table 22 in Example 6, and Table 38 in Example 9) include a methionine residue at the N-terminal end of the amino acid sequence. However, with respect to the present disclosure and for the purpose of identifying and numbering residues in the MMLV RTase amino acid sequence where mutations have been introduced, this methionine residue is considered to be amino acid residue 0 (i.e., is not counted) and the second amino acid residue (e.g., threonine in the MMLV RTase base construct set forth in SEQ ID NO: 637 and SEQ ID NO: 717) is considered to be amino acid residue 1.

TABLE 3 Sequences of MMLV RTase base construct and single mutant MMLV RTase constructs. SEQ ID Construct Sequence NO: Construct (DNA: 5′-3′ or AA) 636 MMLV RTase ATGACTTTAAATATTGAGGA TGAGCATCGTTTACATGAGA CATCAAAAGAACCCGACGTG AGCTTAGGGTCAACGTGGCT TTCTGACTTCCCCCAGGCGT GGGCGGAGACTGGCGGAATG GGGTTAGCTGTCCGCCAAGC ACCGTTGATCATCCCGTTAA AGGCAACGTCTACACCTGTC TCTATCAAACAGTACCCCAT GAGTCAAGAGGCCCGCCTGG GGATTAAGCCACATATTCAG CGCTTGCTGGACCAGGGGAT CTTGGTCCCATGTCAATCTC CGTGGAACACCCCCCTTCTG CCCGTGAAAAAGCCAGGTAC AAACGATTATCGTCCAGTTC AAGATCTTCGCGAGGTCAAC AAACGCGTAGAAGACATCCA TCCGACTGTACCTAATCCTT ATAATCTGTTATCAGGCCTG CCCCCATCGCACCAATGGTA TACAGTATTAGACTTGAAAG ACGCGTTCTTTTGCCTGCGT CTGCACCCAACGTCTCAGCC GCTGTTTGCGTTCGAATGGC GTGATCCTGAAATGGGAATT TCGGGTCAGTTAACCTGGAC TCGTCTGCCCCAGGGCTTTA AAAACAGCCCCACATTGTTC GATGAAGCACTTCACCGTGA CTTAGCAGACTTCCGTATCC AACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCT TTTGTTGGCGGCAACGTCTG AACTTGACTGTCAGCAAGGC ACACGCGCGTTATTACAAAC GTTAGGTAACTTAGGATATC GTGCGTCCGCGAAAAAGGCG CAAATTTGTCAAAAACAGGT AAAGTACCTTGGGTATTTGC TGAAAGAAGGTCAACGTTGG CTGACTGAAGCGCGTAAGGA GACCGTAATGGGGCAGCCTA CGCCTAAGACGCCACGCCAG TTGCGTGAATTTTTGGGCAC AGCGGGATTCTGTCGTTTAT GGATTCCTGGGTTCGCTGAA ATGGCTGCACCCCTGTACCC CTTAACAAAAACAGGGACGC TTTTCAACTGGGGGCCAGAC CAGCAAAAGGCGTATCAGGA GATCAAACAAGCTTTGTTGA CCGCACCCGCGTTGGGTCTT CCGGATTTAACCAAGCCCTT TGAGCTGTTCGTTGATGAAA AACAGGGATATGCAAAAGGA GTATTAACCCAAAAGTTAGG CCCGTGGCGTCGCCCTGTTG CTTACTTGAGTAAAAAATTG GATCCTGTCGCAGCAGGATG GCCACCGTGCTTGCGTATGG TCGCGGCAATTGCCGTTTTG ACAAAGGATGCAGGTAAGTT GACGATGGGTCAACCCTTAG TAATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCA GCCCCCAGACCGCTGGCTTT CTAATGCGCGCATGACCCAC TATCAGGCGCTTCTGCTTGA TACGGATCGTGTACAATTTG GACCAGTTGTAGCTTTGAAT CCAGCTACTTTGCTTCCCCT TCCAGAAGAAGGACTTCAGC ACAATTGTTTAGATATTCTG GCCGAGGCACATGGGACGCG CCCTGATTTGACGGATCAGC CACTGCCTGATGCCGACCAT ACATGGTATACTGGCGGCAG TAGTCTTCTTCAAGAGGGGC AACGCAAGGCGGGAGCAGCC GTCACTACGGAGACCGAAGT TATCTGGGCCAAAGCGTTAC CCGCGGGAACATCCGCGCAA CGTGCACAGTTAATCGCTCT GACACAGGCCCTGAAGATGG CAGAGGGCAAAAAGTTGAAT GTCTACACCAACTCACGTTA TGCTTTTGCAACAGCGCATA TCCATGGCGAAATTTACCGC CGCCGTGGTCTGCTGACTAG TGAGGGTAAGGAAATTAAAA ATAAAGATGAGATTCTTGCG TTGTTAAAAGCTTTATTCTT ACCAAAACGCCTTTCGATCA TTCATTGCCCGGGGCATCAA AAGGGTCACTCAGCGGAGGC TCGTGGAAACCGTATGGCGG ACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGA TACATCAACGCTGTTGATCG AAAACAGCTCTCCCTACACT AGCGAGCATTTTTAA 637 MMLV RTase MTLNIEDEHRLHETSKEPDV SLGSTWLSDFPQAWAETGGM GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPWALNP ATLLPLPEEGLQHNCLDILA EAHGTRPDLTDQPLPDADHT WYTGGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNV YTNSRYAFATAHIHGEIYRR RGLLTSEGKEIKNKDEILAL LKALFLPKRLSIIHCPGHQK GHSAEARGNRMADQAARKAA ITETPDTSTLLIENSSPYTS EHF 638 MMLV RTase MTLNIEDEHRLHETSKEPDV with 161R SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SRKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDL ILLQYVDDLLLAATSELDCQ QGTRALLQTLGNLGYRASAK KAQICQKQVKYLGYLLKEGQ RWLTEARKETVMGQPTPKTP RQLREFLGTAGFCRLWIPGF AEMAAPLYPLTKTGTLFNWG PDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYA KGVLTQKLGPWRRPVAYLSK KLDPVAAGWPPCLRMVAAIA VLTKDAGKLTMGQPLVILAP HAVEALVKQPPDRWLSNARM THYQALLLDTDRVQFGPWAL NPATLLPLPEEGLQHNCLDI LAEAHGTRPDLTDQPLPDAD HTWYTGGSSLLQEGQRKAGA AVTTETEVIWAKALPAGTSA QRAQLIALTQALKMAEGKKL NVYTNSRYAFATAHIHGEIY RRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGH QKGHSAEARGNRMADQAARK AAITETPDTSTLLIENSSPY TSEHF 639 MMLV RTase MTLNIEDEHRLHETSKEPDV with Q68R SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSREARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPWALNP ATLLPLPEEGLQHNCLDILA EAHGTRPDLTDQPLPDADHT WYTGGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNV YTNSRYAFATAHIHGEIYRR RGLLTSEGKEIKNKDEILAL LKALFLPKRLSIIHCPGHQK GHSAEARGNRMADQAARKAA ITETPDTSTLLIENSSPYTS EHF 640 MMLV RTase MTLNIEDEHRLHETSKEPDV with Q79R SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIR RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTK TGTLFNWGPDQQKAYQEIKQ ALLTAPALGLPDLTKPFELF VDEKQGYAKGVLTQKLGPWR RPVAYLSKKLDPVAAGWPPC LRMVAAIAVLTKDAGKLTMG QPLVILAPHAVEALVKQPPD RWLSNARMTHYQALLLDTDR VQFGPWALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLT DQPLPDADHTWYTGGSSLLQ EGQRKAGAAVTTETEVIWAK ALPAGTSAQRAQLIALTQAL KMAEGKKLNVYTNSRYAFAT AHIHGEIYRRRGLLTSEGKE IKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNR MADQAARKAAITETPDTSTL LIENSSPYTSEHF 641 MMLV RTase MTLNIEDEHRLHETSKEPDV with L99R SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPRL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPWALNP ATLLPLPEEGLQHNCLDILA EAHGTRPDLTDQPLPDADHT WYTGGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNV YTNSRYAFATAHIHGEIYRR RGLLTSEGKEIKNKDEILAL LKALFLPKRLSIIHCPGHQK GHSAEARGNRMADQAARKAA ITETPDTSTLLIENSSPYTS EHF 642 MMLV RTase MTLNIEDEHRLHETSKEPDV with E282D SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQ ALLLDTDRVQFGPWALNPAT LLPLPEEGLQHNCLDILAEA HGTRPDLTDQPLPDADHTWY TGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQ LIALTQALKMAEGKKLNVYT NSRYAFATAHIHGEIYRRRG LLTSEGKEIKNKDEILALLK ALFLPKRLSIIHCPGHQKGH SAEARGNRMADQAARKAAIT ETPDTSTLLIENSSPYTSEH F 643 MMLV RTase MTLNIEDEHRLHETSKEPDV with R298A SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPAQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPWALNP ATLLPLPEEGLQHNCLDILA EAHGTRPDLTDQPLPDADHT WYTGGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNV YTNSRYAFATAHIHGEIYRR RGLLTSEGKEIKNKDEILAL LKALFLPKRLSIIHCPGHQK GHSAEARGNRMADQAARKAA ITETPDTSTLLIENSSPYTS EHF e. Experimental Results

The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase single mutant variants were analyzed and reported by copy number output based on a standard curve (see Tables 4 and 5). Six single mutant MMLV RTase variants were found to exhibit an increase in the overall activity and thermostability as compared to the MMLV RTase base construct. The six single mutant MMLV RTase variants were as follows: I61R, Q68R, Q79R, L99R, E282D, and R298A.

TABLE 4 Two-step cDNA synthesis by MMLV RT single mutants. Data was generated via qPCR human normalizer assay and translated by copy number. MMLV RT Variant Quantity Mean Quantity Standard Deviation MMLV-II 21,046.784 954.827 MMLV-II A283V 280.423 50.910 MMLV-II A283R 10,390.819 340.236 MMLV-II A283E 7,378.705 122.716 MMLV-II E123A 15,059.791 556.095 MMLV-II E123R 19,043.292 415.522 MMLV-II E123D 3,619.959 243.766 MMLV-II E282A 19,939.551 1,645.246 MMLV-II E282R 15,588.940 546.467 MMLV-II E282D 24,282.327 2,259.264 MMLV-II I61A 648.252 45.640 MMLV-II I61R 26,280.811 549.417 MMLV-II I61E 10,966.741 469.747 MMLV-II K102A 98.438 12.778 MMLV-II K102R 780.114 90.331 MMLV-II K102E 1,674.854 157.485 MMLV-II K103A 359.984 67.322 MMLV-II K103R 206.765 20.758 MMLV-II K103E 200.883 16.719 MMLV-II K120A 217.787 72.696 MMLV-II K120R 3,619.338 100.478 MMLV-II K120E 2,230.375 210.050 MMLV-II K193A 2,736.271 162.383 MMLV-II K193R 11,496.935 193.681 MMLV-II K193E 325.109 50.932 MMLV-II K295A 8,101.927 348.373 MMLV-II K295R 6,879.112 131.993 MMLV-II K295E 9,673.612 351.106 MMLV-II K329A 3,199.167 212.003 MMLV-II K329R 10,387.670 330.429 MMLV-II K329E 18,306.813 1,167.600 MMLV-II K53A 474.465 62.390 MMLV-II K53R 369.020 49.436 MMLV-II K53E 5,308.165 104.585 MMLV-II K62A 2,102.396 64.197 MMLV-II K62R 4,920.330 251.414 MMLV-II K62E 71.723 11.419 MMLV-II K75A 76.659 24.657 MMLV-II K75R 2,842.314 77.212 MMLV-II K75E 1,697.887 158.946 MMLV-II L99A 1,576.246 213.455 MMLV-II L99R 37,070.048 1,531.910 MMLV-II L99E 195.448 22.530 MMLV-II N107A 3,354.325 176.385 MMLV-II N107R 41.532 24.527 MMLV-II N107E 8,523.285 353.411 MMLV-II Q291A 14,093.444 576.318 MMLV-II Q291R 15,736.443 566.630 MMLV-II Q291E 1,480.309 93.187 MMLV-II Q68A n.d. n.d. MMLV-II Q68R 20,158.035 722.022 MMLV-II Q68E 2,263.714 150.236 MMLV-II Q79A 2,317.484 43.518 MMLV-II Q79R 37,480.443 1,268.309 MMLV-II Q79E 489.184 39.449 MMLV-II R110A 1,815.710 7.917 MMLV-II R110K 502.172 38.619 MMLV-II R110E 383.331 38.162 MMLV-II R298A 44,477.013 3,036.502 MMLV-II R298K 14,925.202 186.581 MMLV-II R298E 1,150.932 56.107 MMLV-II R301A 2,745.075 82.646 MMLV-II R301K 12,813.899 568.898 MMLV-II R301E 1,583.826 198.913 MMLV-II T106A 16,641.642 179.631 MMLV-II T106R 2,248.217 71.295 MMLV-II T106E 10,302.113 250.531 MMLV-II T128V 7,034.032 351.446 MMLV-II T128R 3,465.069 143.456 MMLV-II T128E 10,709.019 110.124 MMLV-II T293A 4,612.880 167.335 MMLV-II T293R 13,753.879 319.851 MMLV-II T293E 12,893.457 223.100 MMLV-II T296A 2,192.531 76.071 MMLV-II T296R 893.449 51.913 MMLV-II T296E 473.936 102.414 MMLV-II T55A 5,774.471 223.173 MMLV-II T55R 3,284.089 314.651 MMLV-II T55E 6,143.058 429.507 MMLV-II T57A 6,129.791 285.070 MMLV-II T57R 888.244 11.952 MMLV-II T57E 1,487.448 71.681 MMLV-II V101A 552.130 98.391 MMLV-II V101R 4,754.017 107.434 MMLV-II V101E 1,388.699 87.091 MMLV-II V112A 2,085.594 72.265 MMLV-II V112R 377.194 41.722 MMLV-II V112E 210.825 17.715 MMLV-II V59A 628.779 15.216 MMLV-II V59R 6,662.173 210.234 MMLV-II V59E 3,249.465 79.848 MMLV-II Y109A 101.656 6.717 MMLV-II Y109R 349.373 27.171 MMLV-II Y109E 1,029.589 45.189 MMLV-IV 71,572.714 4,656.679

TABLE 5 One-step cDNA synthesis by MMLV RT single mutants. Data was generated via qPCR human normalizer assay and data is translated by copy number. MMLV RT Variant Quantity Mean Quantity Standard Deviation MMLV-II 20,638.973 614.785 MMLV-II A283V 8,802.753 220.902 MMLV-II A283R 14,379.575 337.562 MMLV-II A283E 16,396.614 203.476 MMLV-II E123A 17,975.218 259.986 MMLV-II E123R 20,652.508 515.600 MMLV-II E123D 14,452.672 242.000 MMLV-II E282A 19,017.751 827.419 MMLV-II E282R 17,180.421 204.739 MMLV-II E282D 20,735.271 420.881 MMLV-II I61A 7,450.147 348.788 MMLV-II I61R 25,123.507 2,977.836 MMLV-II I61E 17,441.860 1,662.749 MMLV-II K102A 9,342.754 120.846 MMLV-II K102R 10,563.589 255.139 MMLV-II K102E 13,925.008 307.601 MMLV-II K103A 9,429.555 437.351 MMLV-II K103R 9,009.846 155.888 MMLV-II K103E 7,985.278 189.792 MMLV-II K120A 8,593.433 438.722 MMLV-II K120R 12,558.793 407.946 MMLV-II K120E 12,268.574 303.495 MMLV-II K193A 12,977.263 537.992 MMLV-II K193R 13,446.766 2,337.906 MMLV-II K193E 8,536.558 182.514 MMLV-II K295A 13,506.491 1,613.467 MMLV-II K295R 13,944.407 1,839.608 MMLV-II K295E 15,021.823 650.111 MMLV-II K329A 13,284.541 246.298 MMLV-II K329R 15,935.899 970.971 MMLV-II K329E 20,628.859 884.254 MMLV-II K53A 10,868.676 161.435 MMLV-II K53R 9,908.252 632.663 MMLV-II K53E 20,666.775 518.895 MMLV-II K62A 9,454.043 732.242 MMLV-II K62R 14,532.171 63.450 MMLV-II K62E 8,341.361 436.076 MMLV-II K75A 9,084.502 113.100 MMLV-II K75R 13,106.462 331.663 MMLV-II K75E 11,191.849 565.160 MMLV-II L99A 12,876.076 49.507 MMLV-II L99R 27,167.197 142.371 MMLV-II L99E 6,534.199 2,730.598 MMLV-II N107A 13,563.421 349.378 MMLV-II N107R 8,654.167 497.167 MMLV-II N107E 16,675.075 172.596 MMLV-II Q291A 20,957.729 150.006 MMLV-II Q291R 17,980.723 346.436 MMLV-II Q291E 11,025.722 407.116 MMLV-II Q68A n.d. n.d. MMLV-II Q68R 24,925.791 937.265 MMLV-II Q68E 12,844.484 165.039 MMLV-II Q79A 12,038.975 482.596 MMLV-II Q79R 28,458.521 296.595 MMLV-II Q79E 10,358.863 309.043 MMLV-II R110A 11,517.764 562.094 MMLV-II R110K 8,112.167 76.742 MMLV-II R110E 8,809.423 290.785 MMLV-II R298A 27,817.905 172.690 MMLV-II R298K 18,222.660 825.743 MMLV-II R298E 10,783.790 783.279 MMLV-II R301A 11,344.854 63.499 MMLV-II R301K 17,584.850 445.587 MMLV-II R301E 10,146.906 1,879.902 MMLV-II T106A 17,717.520 215.965 MMLV-II T106R 11,680.187 148.213 MMLV-II T106E 21,203.557 366.469 MMLV-II T128V 14,384.970 355.754 MMLV-II T128R 12,938.223 464.841 MMLV-II T128E 14,781.394 1,930.931 MMLV-II T293A 15,658.189 347.640 MMLV-II T293R 19,976.165 253.604 MMLV-II T293E 17,580.335 404.397 MMLV-II T296A 10,312.142 159.775 MMLV-II T296R 8,482.071 92.806 MMLV-II T296E 7,687.972 112.884 MMLV-II T55A 18,073.262 618.174 MMLV-II T55R 11,546.179 138.906 MMLV-II T55E 12,299.658 815.911 MMLV-II T57A 14,700.042 2,916.521 MMLV-II T57R 11,195.901 145.433 MMLV-II T57E 11,958.503 605.445 MMLV-II V101A 10,697.751 269.696 MMLV-II VI01R 8,934.765 53.924 MMLV-II V101E 11,295.874 296.506 MMLV-II V112A 12,854.738 356.724 MMLV-II V112R 6,331.802 303.453 MMLV-II V112E 7,643.184 448.446 MMLV-II V59A 9,520.143 339.954 MMLV-II V59R 18,523.053 499.377 MMLV-II V59E 16,029.631 137.454 MMLV-II Y109A 8,421.361 185.196 MMLV-II Y109R 8,581.961 129.732 MMLV-II Y109E 10,216.473 416.388 MMLV-IV 65,726.159 1,811.314

Example 4: Extension of Reverse Transcriptase Single Mutants

The amino acid positions that enclosed the MMLV RTase single mutants identified in Example 3 were further evaluated to include all possible amino acid substitutions at that position. The single mutants were cloned, overexpressed, and purified as described in Examples 1 and 2, and evaluated as described in Example 3. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase double mutant variants were analyzed and reported by copy number output based on a standard curve (see Tables 6 and 7). Ten single mutant MMLV RTase variants (see Table 8) were found to exhibit an increase in the overall activity and thermostability as compared to the MMLV RTase base construct. The ten single mutant MMLV RTase variants were as follows: I61K, I61M, Q68I, Q68K, Q79H, Q79I, L99K, L99N, E282M and E282W.

TABLE 6 Two-step cDNA synthesis by MMLV RT single mutants. Data was generated via qPCR human normalizer assay and translated by copy number. MMLV RT Variant Quantity Mean Quantity Standard Deviation MMLV-II 1,484.121 125.278 MMLV-II E282C 749.332 37.947 MMLV-II E282F 968.042 28.112 MMLV-II E282G 841.839 30.618 MMLV-II E282H 936.562 64.904 MMLV-II E282I 1,418.551 8.682 MMLV-II E282K 2,399.973 50.862 MMLV-II E282L 1,778.903 134.133 MMLV-II E282M 2,115.328 125.477 MMLV-II E282N 1,175.130 79.221 MMLV-II E282P 1,529.331 61.525 MMLV-II E282Q 1,856.418 24.118 MMLV-II E282S 673.670 44.770 MMLV-II E282T 994.318 24.066 MMLV-II E282V 748.877 29.053 MMLV-II E282W 2,469.404 141.080 MMLV-II E282Y 1,360.706 338.309 MMLV-II I61C 283.240 11.244 MMLV-II I61D 349.008 10.979 MMLV-II I61F 784.163 22.643 MMLV-II I61G 395.348 21.967 MMLV-II I61H 736.015 30.271 MMLV-II I61K 4,479.606 62.627 MMLV-II I61L 1,106.547 38.553 MMLV-II I61M 4,198.088 93.025 MMLV-II I61N 709.752 29.312 MMLV-II I61P 32.935 16.814 MMLV-II I61Q 1,311.695 145.810 MMLV-II I61S 797.783 50.626 MMLV-II I61T 628.173 33.371 MMLV-II I61V 1,439.915 27.490 MMLV-II I61W 442.039 29.310 MMLV-II I61Y 534.249 26.831 MMLV-II L99C 3,109.142 80.016 MMLV-II L99D 83.653 3.432 MMLV-II L99F 2,811.513 79.584 MMLV-II L99G 908.041 16.157 MMLV-II L99H 4,881.196 390.497 MMLV-II L99I 910.072 71.671 MMLV-II L99K 6,410.818 127.262 MMLV-II L99M 976.548 65.154 MMLV-II L99N 4,974.458 162.464 MMLV-II L99P 6.416 1.820 MMLV-II L99Q 3,908.473 337.167 MMLV-II L99S 3,793.955 86.959 MMLV-II L99T 4,189.211 27.640 MMLV-II L99V 964.081 48.105 MMLV-II L99W 1,614.660 40.442 MMLV-II L99Y 2,123.406 181.945 MMLV-II Q68A 1,184.702 7.676 MMLV-II Q68C 2,038.167 36.463 MMLV-II Q68D 1,613.880 77.796 MMLV-II Q68F 1,805.647 62.456 MMLV-II Q68G 2,262.873 69.688 MMLV-II Q68H 106.421 9.860 MMLV-II Q68I 2,675.446 73.874 MMLV-II Q68K 1,042.979 70.081 MMLV-II Q68L 1,070.742 57.215 MMLV-II Q68M 1,342.806 58.349 MMLV-II Q68N 1,993.946 65.808 MMLV-II Q68P 2,025.753 25.540 MMLV-II Q68S 1,895.984 26.959 MMLV-II Q68T 431.442 22.751 MMLV-II Q68V 1,534.710 110.794 MMLV-II Q68W 1,790.706 124.583 MMLV-II Q79C 2,477.812 107.510 MMLV-II Q79D 627.902 11.073 MMLV-II Q79F 1,786.571 126.904 MMLV-II Q79G 2,702.985 83.998 MMLV-II Q79H 2,851.710 57.501 MMLV-II Q79I 2,967.710 57.440 MMLV-II Q79K 1,346.751 64.513 MMLV-II Q79L 2,214.615 67.622 MMLV-II Q79M 1,847.181 31.384 MMLV-II Q79N 1,365.563 54.775 MMLV-II Q79P 674.074 42.100 MMLV-II Q79S 2,199.353 52.958 MMLV-II Q79T 1,523.163 77.025 MMLV-II Q79V 1,704.661 77.643 MMLV-II Q79W 2,186.489 31.470 MMLV-II Q79Y 2,326.023 123.508 MMLV-II R298C 79.970 9.815 MMLV-II R298D 0.000 0.000 MMLV-II R298F 84.760 9.362 MMLV-II R298G 357.027 15.726 MMLV-II R298H 269.257 20.814 MMLV-II R298I 130.983 5.364 MMLV-II R298L 199.612 5.843 MMLV-II R298M 172.013 18.710 MMLV-II R298N 199.678 2.660 MMLV-II R298P 122.098 5.900 MMLV-II R298Q 118.092 40.694 MMLV-II R298S 406.112 7.695 MMLV-II R298T 618.616 20.023 MMLV-II R298V 136.498 13.297 MMLV-II R298W 68.096 7.016 MMLV-II R298Y 162.713 7.854 MMLV-IV 6,830.294 376.878

TABLE 7 One-step cDNA synthesis by MMLV RT single mutants. Data was generated via qPCR human normalizer assay and data is translated by copy number. MMLV RT Variant Quantity Mean Quantity Standard Deviation MMLV-II 408.018 8.693 MMLV-II E282C 175.083 7.005 MMLV-II E282F 1,043.025 16.137 MMLV-II E282G 635.037 13.293 MMLV-II E282H 656.956 10.018 MMLV-II E282I 1,033.125 44.996 MMLV-II E282K 751.309 17.611 MMLV-II E282L 1,072.350 80.365 MMLV-II E282M 1,318.072 51.735 MMLV-II E282N 539.305 10.767 MMLV-II E282P 725.869 92.685 MMLV-II E282Q 626.674 12.129 MMLV-II E282S 354.956 34.850 MMLV-II E282T 485.477 45.783 MMLV-II E282V 594.047 27.898 MMLV-II E282W 913.290 61.145 MMLV-II E282Y 759.920 34.784 MMLV-II I61C 219.438 18.403 MMLV-II I61D 347.020 13.303 MMLV-II I61F 428.623 25.316 MMLV-II I61G 389.503 21.764 MMLV-II I61H 514.330 18.416 MMLV-II I61K 2,343.894 67.214 MMLV-II I61L 621.572 14.892 MMLV-II I61M 2,536.807 150.371 MMLV-II I61N 538.519 20.736 MMLV-II I61P 61.683 18.802 MMLV-II I61Q 701.471 32.487 MMLV-II I61S 611.977 30.430 MMLV-II I61T 534.254 31.643 MMLV-II I61V 881.608 20.662 MMLV-II I61W 428.440 17.964 MMLV-II I61Y 347.930 4.412 MMLV-II L99C 2,390.104 35.867 MMLV-II L99D 185.044 6.975 MMLV-II L99F 1,577.767 7.757 MMLV-II L99G 987.225 9.718 MMLV-II L99H 3,886.372 111.670 MMLV-II L99I 613.648 46.303 MMLV-II L99K 7,597.650 321.753 MMLV-II L99M 934.817 52.006 MMLV-II L99N 4,689.222 160.641 MMLV-II L99P 18.537 1.131 MMLV-II L99Q 2,394.744 64.077 MMLV-II L99S 3,293.831 111.802 MMLV-II L99T 3,505.113 101.670 MMLV-II L99V 677.756 49.356 MMLV-II L99W 839.088 50.301 MMLV-II L99Y 1,127.536 19.074 MMLV-II Q68A 827.617 30.689 MMLV-II Q68C 1,110.680 45.944 MMLV-II Q68D 1,045.802 25.488 MMLV-II Q68F 1,210.166 120.899 MMLV-II Q68G 907.279 30.688 MMLV-II Q68H 150.384 6.867 MMLV-II Q68I 1,550.372 76.712 MMLV-II Q68K 1,712.176 47.342 MMLV-II Q68L 651.039 51.426 MMLV-II Q68M 1,395.463 34.805 MMLV-II Q68N 1,241.364 25.780 MMLV-II Q68P 1,249.444 13.709 MMLV-II Q68S 1,125.260 21.324 MMLV-II Q68T 792.901 31.513 MMLV-II Q68V 1,026.654 24.972 MMLV-II Q68W 1,594.175 101.221 MMLV-II Q79C 1,948.151 87.341 MMLV-II Q79D 458.131 10.763 MMLV-II Q79F 1,623.675 50.723 MMLV-II Q79G 1,885.097 20.190 MMLV-II Q79H 2,508.763 149.926 MMLV-II Q79I 2,329.030 76.545 MMLV-II Q79K 1,861.302 24.320 MMLV-II Q79L 1,496.247 30.399 MMLV-II Q79M 1,496.469 38.178 MMLV-II Q79N 995.813 42.279 MMLV-II Q79P 526.914 23.216 MMLV-II Q79S 1,685.124 42.694 MMLV-II Q79T 966.505 8.377 MMLV-II Q79V 1,218.191 21.512 MMLV-II Q79W 1,962.326 37.135 MMLV-II Q79Y 2,218.504 56.938 MMLV-II R298C 45.500 1.456 MMLV-II R298D 0.000 0.000 MMLV-II R298F 104.825 5.133 MMLV-II R298G 323.542 14.052 MMLV-II R298H 253.202 47.711 MMLV-II R298I 205.982 8.304 MMLV-II R298L 213.674 15.199 MMLV-II R298M 176.347 12.484 MMLV-II R298N 142.969 39.198 MMLV-II R298P 188.995 3.689 MMLV-II R298Q 95.525 44.292 MMLV-II R298S 307.614 9.962 MMLV-II R298T 487.828 3.480 MMLV-II R298V 255.828 12.902 MMLV-II R298W 37.872 8.482 MMLV-II R298Y 153.333 25.137 MMLV-IV 19,407.721 466.310

TABLE 8 Sequences of single mutant MMLV RTase variants. SEQ ID NO: Construct Construct Sequence (AA) 644 MMLV RTase MTLNIEDEHRLHETSKEPDV with 161K SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SKKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 645 MMLV RTase MTLNIEDEHRLHETSKEPDV with 161M SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SMKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 646 MMLV RTase MTLNIEDEHRLHETSKEPDV with Q68I SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSIEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 647 MMLV RTase MTLNIEDEHRLHETSKEPDV with Q68K SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSKEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 648 MMLV RTase MTLNIEDEHRLHETSKEPDV with Q79H SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIH RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 649 MMLV RTase MTLNIEDEHRLHETSKEPDV with Q79I SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHII RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 650 MMLV RTase MTLNIEDEHRLHETSKEPDV with L99K SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTP VSIKQYPMSQEA RLGIKPHIQRLLDQGILVPC QSPWNTPLKPVKKPGTNDYR PVQDLREVNKRVEDIHPTVP NPYNLLSGLPPSHQWYTVLD LKDAFFCLRLHPTSQPLFAF EWRDPEMGISGQLTWTRLPQ GFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNL GYRASAKKAQICQKQVKYLG YLLKEGQRWLTEARKETVMG QPTPKTPRQLREFLGTAGFC RLWIPGFAEMAAPLYPLTKT GTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFV DEKQGYAKGVLTQKLGPWRR PVAYLSKKLDPVAAGWPPCL RMVAAIAVLTKDAGKLTMGQ PLVILAPHAVEALVKQPPDR WLSNARMTHYQALLLDTDRV QFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLT DQPLPDADHTWYTGGSSLLQ EGQRKAGAAVTTETEVIWAK ALPAGTSAQRAQLIALTQAL KMAEGKKLNVYTNSRYAFAT AHIHGEIYRRRGLLTSEGKE IKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNR MADQAARKAAITETPDTSTL LIENSSPYTSEHF 651 MMLV RTase MTLNIEDEHRLHETSKEPDV with L99N SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLN PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 652 MMLV RTase MTLNIEDEHRLHETSKEPDV with E282M SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTMARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 653 MMLV RTase MTLNIEDEHRLHETSKEPDV with E282W SLGSTWLSDFPQAWAETGGM mutation GLAVRQAPLIIPLKATSTPV SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTWARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF

Example 5: Stacking of Reverse Transcriptase Mutants with Enhanced Activity

a. MMLV RTase Double Mutants

The MMLV RTase single mutants identified in Example 3 were stacked to further improve the ability of MMLV RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) as compared to the MMLV RTase base construct (RNase H minus construct). Fifteen MMLV RTase double mutant variants (see Table 9) were cloned, overexpressed, and purified as described in Examples 1 and 2, and evaluated as described in Example 3. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase double mutant variants were analyzed and reported by copy number output based on a standard curve (see Tables 10 and 11).

Four of the fifteen MMLV RTase double mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase double mutant variants, and almost all of the MMLV RTase double mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The four MMLV RTase double mutant variants that were found to exhibit the highest overall activity were E282D/L99R, L99R/Q68R, L99R/Q79R, and Q68R/Q79R.

TABLE 9 Sequences of double mutant MMLV RTase variants. SEQ ID NO: Construct Construct Sequence (AA) 654 MMLV RTase MTLNIEDEHRLHETSKEPDV With SLGSTWLSDFPQAWAETGGM I61R/E282D GLAVRQAPLIIPLKATSTPV mutations SRKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 655 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM L99R/E282D GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPRL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 656 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/E282D GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSREARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 657 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q79R/E282D GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSQEARLGIKPHIR RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 658 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM E282D/R298A GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPAQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 659 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM I61R/L99R GLAVRQAPLIIPLKATSTPV mutations SRKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLR PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 660 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM I61R/Q68R GLAVRQAPLIIPLKATSTPV mutations SRKQYPMSREARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEH 661 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM I61R/Q79R GLAVRQAPLIIPLKATSTPV mutations SRKQYPMSQEARLGIKPHIR RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 662 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM I61R/R298A GLAVRQAPLIIPLKATSTPV mutations SRKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPAQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 663 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/L99R GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSREARLGIKPHIQ RLLDQGILVPCQSPWNTPLR PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 664 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q79R/L99R GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSQEARLGIKPHIR RLLDQGILVPCQSPWNTPLR PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 665 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM L99R/R298A GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSQEARLGIKPHIQ RLLDQGILVPCQSPWNTPLR PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPAQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 666 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/Q79R GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSREARLGIKPHIR RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 667 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/R298A GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSREARLGIKPHIQ RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPAQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 668 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q79R/R298A GLAVRQAPLIIPLKATSTPV mutations SIKQYPMSQEARLGIKPHIR RLLDQGILVPCQSPWNTPLL PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPAQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTKPFELF VDEKQGYAKGVLTQKLGPWR RPVAYLSKKLDPVAAGWPPC LRMVAAIAVLTKDAGKLTMG QPLVILAPHAVEALVKQPPD RWLSNARMTHYQALLLDTDR VQFGPVVALNPATLLPLPEE GLQHNCLDILAEAHGTRPDL TDQPLPDADHTWYTGGSSLL QEGQRKAGAAVTTETEVIWA KALPAGTSAQRAQLIALTQA LKMAEGKKLNVYTNSRYAFA TAHIHGEIYRRRGLLTSEGK EIKNKDEILALLKALFLPKR LSIIHCPGHQKGHSAEARGN RMADQAARKAAITETPDTST LLIENSSPYTSEHFTAPALG LPDL

TABLE 10 Two-Step cDNA synthesis by MMLV RT double mutants. Data was generated via qPCR human normalizer assay and data is translated by copy number. MMLV RT Variant Quantity Mean Quantity Standard Deviation MMLV-II 1,773.623 5.057 MMLV-II E282D/I61R 4,810.277 143.422 MMLV-II E282D/L99R 7,266.281 50.730 MMLV-II E282D/Q68R 5,186.392 69.563 MMLV-II E282D/Q79R 4,311.403 95.402 MMLV-II E282D/R298A 1,366.524 16.429 MMLV-II I61R/L99R 6,061.812 174.619 MMLV-II I61R/Q68R 5,899.316 39.879 MMLV-II I61R/Q79R 5,257.089 98.378 MMLV-II I61R/R298A 2,661.223 68.948 MMLV-II L99R/Q68R 7,750.519 94.408 MMLV-II L99R/Q79R 7,455.203 124.095 MMLV-II L99R/R298A 5,351.021 179.558 MMLV-II Q68R/Q79R 7,178.681 86.595 MMLV-II Q68R/R298A 4,524.340 84.703 MMLV-II Q79R/R298A 3,739.608 58.621 MMLV-IV 8,258.715 79.458

TABLE 11 One-Step cDNA synthesis by MMLV RT double mutants. Data was generated via qPCR human normalizer assay and data is translated by cony number. MMLV-RT Variant Quantity Mean Quantity Standard Deviation MMLV-II 859.127 24.795 MMLV-II E282D/I61R 2,948.906 49.177 MMLV-II E282D/L99R 4,814.957 239.110 MMLV-II E282D/Q68R 3,709.046 131.434 MMLV-II E282D/Q79R 3,694.187 98.772 MMLV-II E282D/R298A 794.643 39.913 MMLV-II I61R/L99R 3,443.713 180.210 MMLV-II I61R/Q68R 3,525.138 112.288 MMLV-II I61R/Q79R 3,125.990 120.996 MMLV-II I61R/R298A 2,006.208 83.559 MMLV-II L99R/Q68R 6,755.852 102.788 MMLV-II L99R/Q79R 6,709.502 35.997 MMLV-II L99R/R298A 2,128.451 55.565 MMLV-II Q68R/Q79R 6,343.821 140.779 MMLV-II Q68R/R298A 2,406.470 74.117 MMLV-II Q79R/R298A 2,301.759 22.849 MMLV-IV 15,411.857 333.388 b. Cloning of MMLV RTase Triple and More Mutants

Following the double mutant variants, MMLV RTase single mutants were stacked further to improve the ability of MMLV RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) as compared to the MMLV RTase base construct (RNase H minus construct). Seventeen MMLV RTase triple or more mutant variants (see Table 12) were cloned as described in Example 1.

TABLE 12 Sequences of triple or more mutant MMLV RTase variants. SEQ ID Construct Sequence NO: Construct (AA) 669 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV L99R/ SIKQYPMSREARLGIKPHIQ E282D RLLDQGILVPCQSPWNTPLR mutations PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 670 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q79R/ GLAVRQAPLIIPLKATSTPV L99R/ SIKQYPMSQEARLGIKPHIR E282D RLLDQGILVPCQSPWNTPLR mutations PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 671 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR E282D RLLDQGILVPCQSPWNTPLL mutations PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 672 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR L99R RLLDQGILVPCQSPWNTPLR mutations PVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 673 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR L99R/ RLLDQGILVPCQSPWNTPLR E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 674 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR L99K/ RLLDQGILVPCQSPWNTPLK E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 675 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR L99N/ RLLDQGILVPCQSPWNTPLN E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 676 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68I/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSIEARLGIKPHIR L99R/ RLLDQGILVPCQSPWNTPLR E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 677 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68K/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSKEARLGIKPHIR L99R/ RLLDQGILVPCQSPWNTPLR E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 678 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79H/ SIKQYPMSREARLGIKPHIH L99R/ RLLDQGILVPCQSPWNTPLR E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 679 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79I/ SIKQYPMSREARLGIKPHII L99R/ RLLDQGILVPCQSPWNTPLR E282D PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 680 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR L99R/ RLLDQGILVPCQSPWNTPLR E282M PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTMARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 681 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68R/ GLAVRQAPLIIPLKATSTPV Q79R/ SIKQYPMSREARLGIKPHIR L99R/ RLLDQGILVPCQSPWNTPLR E282W PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTWARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 682 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM 161K/ GLAVRQAPLIIPLKATSTPV Q68R/ SKKQYPMSREARLGIKPHIR Q79R/ RLLDQGILVPCQSPWNTPLR L99R/ PVKKPGTNDYRPVQDLREVN E282D KRVEDIHPTVPNPYNLLSGL mutations PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 683 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM 161M/ GLAVRQAPLIIPLKATSTPV Q68R/ SMKQYPMSREARLGIKPHIR Q79R/ RLLDQGILVPCQSPWNTPLR L99R/ PVKKPGTNDYRPVQDLREVN E282D KRVEDIHPTVPNPYNLLSGL mutations PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTDARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDL TKPFELFVDEKQGYAKGVLT QKLGPWRRPVAYLSKKLDPV AAGWPPCLRMVAAIAVLTKD AGKLTMGQPLVILAPHAVEA LVKQPPDRWLSNARMTHYQA LLLDTDRVQFGPVVALNPAT LLPLPEEGLQHNCLDILAEA HGTRPDLTDQPLPDADHTWY TGGSSLLQEGQRKAGAAVTT ETEVIWAKALPAGTSAQRAQ LIALTQALKMAEGKKLNVYT NSRYAFATAHIHGEIYRRRG LLTSEGKEIKNKDEILALLK ALFLPKRLSIIHCPGHQKGH SAEARGNRMADQAARKAAIT ETPDTSTLLIENSSPYTSEH F 684 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM Q68I/ GLAVRQAPLIIPLKATSTPV Q79H/ SIKQYPMSIEARLGIKPHIH L99K/ RLLDQGILVPCQSPWNTPLK E282M PVKKPGTNDYRPVQDLREVN mutations KRVEDIHPTVPNPYNLLSGL PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTMARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF 685 MMLV RTase MTLNIEDEHRLHETSKEPDV with SLGSTWLSDFPQAWAETGGM I61M/ GLAVRQAPLIIPLKATSTPV Q68I/ SMKQYPMSIEARLGIKPHIH Q79H/ RLLDQGILVPCQSPWNTPLK L99K/ PVKKPGTNDYRPVQDLREVN E282M KRVEDIHPTVPNPYNLLSGL mutations PPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLF DEALHRDLADFRIQHPDLIL LQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKA QICQKQVKYLGYLLKEGQRW LTMARKETVMGQPTPKTPRQ LREFLGTAGFCRLWIPGFAE MAAPLYPLTKTGTLFNWGPD QQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKG VLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALN PATLLPLPEEGLQHNCLDIL AEAHGTRPDLTDQPLPDADH TWYTGGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQ RAQLIALTQALKMAEGKKLN VYTNSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYT SEHF c. Expression and Purification of MMLV RTase and Mutant Variants

A colony with the appropriate strain was used to inoculate TB media (200 mL) with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was achieved followed by cooling of the flask for 30 minutes at 4° C. Protein expression was induced by the addition of 1 M IPTG (100 μL), followed by growth at 18° C. for 21 hours. Cells were harvested by spinning samples at 4,700×g for 10 minutes.

Cell pellets were re-suspended in a lysis buffer (50 mM NaPO₄, pH 7.8, 5% glycerol, 300 mM NaCl, 10 mM imidazole, 5 mM DTT, 0.01% n-ocyl-β-D-glucopyranoside, DNaseI, 10 mM CaCl₂), lysozyme (1 mg/mL), and protease inhibitor). The sample was lysed on an Avestin Emulsiflex C3 pre-chilled to 4° C. at 15-20 kpsi with three passes. Cell debris was removed by centrifuging the lysate at 16,000×g for 30 minutes at 4° C.

Cleared lysates were applied to a HisTrap HP column (Cytiva Life Sciences, Cat #17524701). The resin was equilibrated with MMLV His-Bind buffer (50 mM NaPO₄, pH 7.8, 5% glycerol, 0.3 M NaCl, 10 mM imidazole, 1 mM DTT and 0.01% IGEPAL-CA), followed by sample loading. The samples were washed with MMLV His-Bind buffer, followed by a 25% B wash (B=MMLV His Elution buffer=50 mM NaPO₄, pH 7.8, 5% glycerol, 0.3 M NaCl, 250 mM imidazole, 1 mM DTT and 0.01% IGEPAL-CA). The sample was eluted with 100% B for 10 CVs in 45 mL fractions.

Purified proteins were applied to a HiTrap Heparin HP column (Cytiva Life Sciences, Cat #17040601). The resin was equilibrated with MMLV Heparin-Bind buffer (50 mM Tris HCl pH 8.5, 75 mM NaCl, 1 mM DTT, 5% glycerol and 0.01% IGEPAL-CA), followed by sample loading. The sample was washed with MLV Heparin Bind buffer, followed by a 25% B wash (B=MLV Heparin Elution Buffer). The sample was eluted with 60% B for 10 CVs in 45 mL fractions.

Purified proteins were applied to a Bio-Scale™ Mini CHT™ Cartridge (Bio-Rad Laboratories, Cat #7324322). The resin was washed with 1 M NaOH, followed by equilibration with MMLV Heparin-Bind buffer and sample loading. The sample was washed with MLV Heparin Elution buffer, followed by MMLV Heparin Bind buffer. The sample was linearly eluted to 100% B2 (B2=MMLV HA Elution Buffer=250 mM KPO4 pH 7.5, 1 mM DTT, 5% glycerol and 0.01% IGEPAL-CA) for 15 CVs in 5 mL fractions.

Fractions containing purified protein were pooled and dialyzed in MMLV Storage Buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 50% (v/v) glycerol).

d. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA by Gene Specific Priming

MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Temperatures were adjusted for both two-step and one-step reactions to 55 and 60° C., respectively. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see Tables 13 and 14).

Six of the seventeen MMLV RTase triple or more mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase stacked mutant variants, and almost all of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The six MMLV RTase mutant variants that were found to exhibit the highest overall activity were Q68R/L99R, Q68R/Q79R/L99R, Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99K/E282D, Q68R/Q79R/L99R/E282W, I61M/Q68R/Q79R/L99R/E282D and Q68I/Q79H/L99K/E282M.

TABLE 13 Two-Step cDNA synthesis by MMLV RT triple and more mutants. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Concentration Ct Ct Standard MMLV RT Variant of RTase (nM) Mean Deviation MMLV-II 0.625 25.520 0.047 MMLV-II L99R/E282D 0.625 24.332 0.060 MMLV-II Q68R/L99R 0.625 22.207 0.097 MMLV-II Q79R/L99R 0.625 23.789 0.012 MMLV-II Q68R/Q79R 0.625 23.629 0.038 MMLV-II Q68R/L99R/E282D 0.625 22.855 0.079 MMLV-II Q79R/L99R/E282D 0.625 23.095 0.035 MMLV-II Q68R/Q79R/E282D 0.625 22.526 0.027 MMLV-II Q68R/Q79R/L99R 0.625 22.099 0.018 MMLV-II 0.625 21.056 0.023 Q68R/Q79R/L99R/E282D MMLV-II 0.625 21.833 0.031 Q68R/Q79R/L99K/E282D MMLV-II 0.625 23.607 0.031 Q68R/Q79R/L99N/E282D MMLV-II 0.625 23.858 0.029 Q68I/Q79R/L99R/E282D MMLV-II 0.625 22.615 0.054 Q68K/Q79R/L99R/E282D MMLV-II 0.625 28.866 0.008 Q68R/Q79H/L99R/E282D MMLV-II 0.625 23.283 0.085 Q68R/Q79I/L99R/E282D MMLV-II 0.625 25.073 0.097 Q68R/Q79R/L99R/E282M MMLV-II 0.625 22.331 0.048 Q68R/Q79R/L99R/E282W MMLV-II 0.625 23.271 0.065 I61K/Q68R/Q79R/L99R/E282D MMLV-II 0.625 22.133 0.018 I61M/Q68R/Q79R/L99R/E282D MMLV-II 0.625 23.344 0.037 Q68I/Q79H/L99K/E282M MMLV-II 0.625 25.255 0.058 I61M/Q68I/Q79H/L99K/E282M MMLV-II 2.5 22.154 0.052 MMLV-II L99R/E282D 2.5 21.501 0.054 MMLV-II Q68R/L99R 2.5 21.151 0.048 MMLV-II Q79R/L99R 2.5 21.229 0.163 MMLV-II Q68R/Q79R 2.5 21.228 0.054 MMLV-II Q68R/L99R/E282D 2.5 21.126 0.030 MMLV-II Q79R/L99R/E282D 2.5 21.418 0.033 MMLV-II Q68R/Q79R/E282D 2.5 21.011 0.052 MMLV-II Q68R/Q79R/L99R 2.5 20.953 0.041 MMLV-II 2.5 21.113 0.108 Q68R/Q79R/L99R/E282D MMLV-II 2.5 20.906 0.081 Q68R/Q79R/L99K/E282D MMLV-II 2.5 21.196 0.029 Q68R/Q79R/L99N/E282D MMLV-II 2.5 21.369 0.009 Q68I/Q79R/L99R/E282D MMLV-II 2.5 20.960 0.030 Q68K/Q79R/L99R/E282D MMLV-II 2.5 26.167 0.038 Q68R/Q79H/L99R/E282D MMLV-II 2.5 21.012 0.056 Q68R/Q79I/L99R/E282D MMLV-II 2.5 21.277 0.036 Q68R/Q79R/L99R/E282M MMLV-II 2.5 20.944 0.020 Q68R/Q79R/L99R/E282W MMLV-II 2.5 21.320 0.009 I61K/Q68R/Q79R/L99R/E282D MMLV-II 2.5 21.095 0.013 I61M/Q68R/Q79R/L99R/E282D MMLV-II 2.5 21.329 0.047 Q68I/Q79H/L99K/E282M MMLV-II 2.5 22.159 0.031 I61M/Q68I/Q79H/L99K/E282M MMLV-II 10 21.575 0.101 MMLV-II L99R/E282D 10 21.546 0.041 MMLV-II Q68R/L99R 10 21.343 0.021 MMLV-II Q79R/L99R 10 21.387 0.016 MMLV-II Q68R/Q79R 10 21.147 0.032 MMLV-II Q68R/L99R/E282D 10 21.265 0.076 MMLV-II Q79R/L99R/E282D 10 21.250 0.036 MMLV-II Q68R/Q79R/E282D 10 21.135 0.015 MMLV-II Q68R/Q79R/L99R 10 21.051 0.036 MMLV-II 10 21.159 0.065 Q68R/Q79R/L99R/E282D MMLV-II 10 21.056 0.032 Q68R/Q79R/L99K/E282D MMLV-II 10 21.180 0.052 Q68R/Q79R/L99N/E282D MMLV-II 10 21.068 0.069 Q68I/Q79R/L99R/E282D MMLV-II 10 21.065 0.053 Q68K/Q79R/L99R/E282D MMLV-II 10 21.683 0.075 Q68R/Q79H/L99R/E282D MMLV-II 10 21.152 0.064 Q68R/Q79I/L99R/E282D MMLV-II 10 21.029 0.055 Q68R/Q79R/L99R/E282M MMLV-II 10 21.214 0.052 Q68R/Q79R/L99R/E282W MMLV-II 10 21.391 0.051 I61K/Q68R/Q79R/L99R/E282D MMLV-II 10 21.307 0.038 I61M/Q68R/Q79R/L99R/E282D MMLV-II 10 21.583 0.019 Q68I/Q79H/L99K/E282M MMLV-II 10 21.759 0.029 I61M/Q68I/Q79H/L99K/E282M

TABLE 14 One-Step cDNA synthesis by MMLV RT triple and more mutants. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Concentration Ct Ct Standard MMLV RT Variant of RTase (nM) Mean Deviation MMLV-II 0.625 22.153 0.122 MMLV-II L99R/E282D 0.625 21.713 0.111 MMLV-II Q68R/L99R 0.625 21.334 0.167 MMLV-II Q79R/L99R 0.625 21.398 0.069 MMLV-II Q68R/Q79R 0.625 21.546 0.096 MMLV-II Q68R/L99R/E282D 0.625 21.112 0.149 MMLV-II Q79R/L99R/E282D 0.625 21.260 0.104 MMLV-II Q68R/Q79R/E282D 0.625 21.014 0.102 MMLV-II Q68R/Q79R/L99R 0.625 20.338 0.042 MMLV-II 0.625 19.537 0.120 Q68R/Q79R/L99R/E282D MMLV-II 0.625 20.516 0.131 Q68R/Q79R/L99K/E282D MMLV-II 0.625 20.960 0.023 Q68R/Q79R/L99N/E282D MMLV-II 0.625 21.325 0.088 Q68I/Q79R/L99R/E282D MMLV-II 0.625 20.602 0.038 Q68K/Q79R/L99R/E282D MMLV-II 0.625 23.889 0.042 Q68R/Q79H/L99R/E282D MMLV-II 0.625 21.375 0.035 Q68R/Q79I/L99R/E282D MMLV-II 0.625 21.805 0.054 Q68R/Q79R/L99R/E282M MMLV-II 0.625 20.229 0.085 Q68R/Q79R/L99R/E282W MMLV-II 0.625 20.972 0.037 I61K/Q68R/Q79R/L99R/E282D MMLV-II 0.625 20.225 0.042 I61M/Q68R/Q79R/L99R/E282D MMLV-II 0.625 20.578 0.061 Q68I/Q79H/L99K/E282M MMLV-II 0.625 21.107 0.101 I61M/Q68I/Q79H/L99K/E282M MMLV-II 2.5 20.874 0.042 MMLV-II L99R/E282D 2.5 19.679 0.047 MMLV-II Q68R/L99R 2.5 19.152 0.024 MMLV-II Q79R/L99R 2.5 19.202 0.091 MMLV-II Q68R/Q79R 2.5 19.506 0.010 MMLV-II Q68R/L99R/E282D 2.5 19.142 0.060 MMLV-II Q79R/L99R/E282D 2.5 19.301 0.004 MMLV-II Q68R/Q79R/E282D 2.5 19.023 0.041 MMLV-II Q68R/Q79R/L99R 2.5 18.312 0.041 MMLV-II 2.5 17.867 0.099 Q68R/Q79R/L99R/E282D MMLV-II 2.5 18.591 0.036 Q68R/Q79R/L99K/E282D MMLV-II 2.5 19.123 0.097 Q68R/Q79R/L99N/E282D MMLV-II 2.5 19.553 0.076 Q68I/Q79R/L99R/E282D MMLV-II 2.5 18.771 0.113 Q68K/Q79R/L99R/E282D MMLV-II 2.5 21.911 0.048 Q68R/Q79H/L99R/E282D MMLV-II 2.5 19.298 0.146 Q68R/Q79I/L99R/E282D MMLV-II 2.5 19.621 0.027 Q68R/Q79R/L99R/E282M MMLV-II 2.5 18.219 0.103 Q68R/Q79R/L99R/E282W MMLV-II 2.5 18.846 0.056 I61K/Q68R/Q79R/L99R/E282D MMLV-II 2.5 18.500 0.042 I61M/Q68R/Q79R/L99R/E282D MMLV-II 2.5 18.752 0.148 Q68I/Q79H/L99K/E282M MMLV-II 2.5 19.445 0.098 I61M/Q68I/Q79H/L99K/E282M MMLV-II 10 18.239 0.025 MMLV-II L99R/E282D 10 17.293 0.021 MMLV-II Q68R/L99R 10 17.144 0.032 MMLV-II Q79R/L99R 10 17.324 0.016 MMLV-II Q68R/Q79R 10 17.123 0.072 MMLV-II Q68R/L99R/E282D 10 17.082 0.088 MMLV-II Q79R/L99R/E282D 10 17.353 0.068 MMLV-II Q68R/Q79R/E282D 10 17.111 0.036 MMLV-II Q68R/Q79R/L99R 10 16.562 0.101 MMLV-II 10 16.492 0.066 Q68R/Q79R/L99R/E282D MMLV-II 10 17.027 0.054 Q68R/Q79R/L99K/E282D MMLV-II 10 17.335 0.080 Q68R/Q79R/L99N/E282D MMLV-II 10 17.726 0.055 Q68I/Q79R/L99R/E282D MMLV-II 10 17.144 0.140 Q68K/Q79R/L99R/E282D MMLV-II 10 19.772 0.064 Q68R/Q79H/L99R/E282D MMLV-II 10 17.424 0.020 Q68R/Q79I/L99R/E282D MMLV-II 10 17.624 0.014 Q68R/Q79R/L99R/E282M MMLV-II 10 16.629 0.080 Q68R/Q79R/L99R/E282W MMLV-II 10 16.903 0.022 I61K/Q68R/Q79R/L99R/E282D MMLV-II 10 16.803 0.028 I61M/Q68R/Q79R/L99R/E282D MMLV-II 10 16.894 0.056 Q6I/Q79H/L99K/E282M MMLV-II 10 17.509 0.058 I61M/Q68I/Q79H/L99K/E282M e. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA by Oligo-dT or Random Priming

MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Oligo-dT or random hexamer priming conditions were adjusted for the two-step reactions and RTase concentration was normalized to 31 nM. The two-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see Tables 15 and 16).

Nine of the seventeen MMLV RTase triple or more mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase stacked mutant variants, and almost all of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The nine MMLV RTase mutant variants that were found to exhibit the highest overall activity were Q79R/L99R/E282D, Q68R/Q79R/L99R, Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99K/E282D, Q68R/Q79R/L99N/E282D, Q68K/Q79R/L99R/E282D, Q68R/Q79R/L99R/E282M, M61K/Q68R/Q79R/L99R/E282D and 161M/Q68R/Q79R/L99R/E282D.

TABLE 15 Two-Step cDNA synthesis by MMLV RT triple and more mutants by Oligo-dT priming. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature of Ct Ct Standard MMLV RT Variant Reaction (° C.) Mean Deviation MMLV-II 42 25.165 0.057 MMLV-II L99R/E282D 42 25.287 0.062 MMLV-II Q68R/L99R 42 25.026 0.035 MMLV-II Q79R/L99R 42 24.932 0.032 MMLV-II Q68R/Q79R 42 25.002 0.076 MMLV-II Q68R/L99R/E282D 42 24.964 0.068 MMLV-II Q79R/L99R/E282D 42 24.822 0.106 MMLV-II Q68R/Q79R/E282D 42 24.905 0.134 MMLV-II Q68R/Q79R/L99R 42 24.673 0.131 MMLV-II 42 24.523 0.111 Q68R/Q79R/L99R/E282D MMLV-II 42 24.677 0.076 Q68R/Q79R/L99K/E282D MMLV-II 42 24.635 0.087 Q68R/Q79R/L99N/E282D MMLV-II 42 25.010 0.074 Q68I/Q79R/L99R/E282D MMLV-II 42 24.676 0.066 Q68K/Q79R/L99R/E282D MMLV-II 42 28.929 0.021 Q68R/Q79H/L99R/E282D MMLV-II 42 24.932 0.039 Q68R/Q79I/L99R/E282D MMLV-II 42 24.900 0.113 Q68R/Q79R/L99R/E282M MMLV-II 42 24.967 0.091 Q68R/Q79R/L99R/E282W MMLV-II 42 24.597 0.076 I61K/Q68R/Q79R/L99R/E282D MMLV-II 42 24.833 0.007 I61M/Q68R/Q79R/L99R/E282D MMLV-II 42 25.440 0.048 Q68I/Q79H/L99K/E282M MMLV-II 42 25.679 0.050 I61M/Q68I/Q79H/L99K/E282M MMLV-II 55 34.223 0.406 MMLV-II L99R/E282D 55 34.732 3.729 MMLV-II Q68R/L99R 55 31.509 0.169 MMLV-II Q79R/L99R 55 31.831 0.019 MMLV-II Q68R/Q79R 55 32.633 1.094 MMLV-II Q68R/L99R/E282D 55 32.089 0.075 MMLV-II Q79R/L99R/E282D 55 32.134 0.081 MMLV-II Q68R/Q79R/E282D 55 34.639 3.791 MMLV-II Q68R/Q79R/L99R 55 29.559 0.029 MMLV-II 55 28.013 0.136 Q68R/Q79R/L99R/E282D MMLV-II 55 29.712 0.090 Q68R/Q79R/L99K/E282D MMLV-II 55 30.442 0.224 Q68R/Q79R/L99N/E282D MMLV-II 55 32.857 0.378 Q68I/Q79R/L99R/E282D MMLV-II 55 31.186 0.630 Q68K/Q79R/L99R/E282D MMLV-II 55 37.338 1.882 Q68R/Q79H/L99R/E282D MMLV-II 55 31.830 0.120 Q68R/Q79I/L99R/E282D MMLV-II 55 31.682 0.181 Q68R/Q79R/L99R/E282M MMLV-II 55 32.256 0.228 Q68R/Q79R/L99R/E282W MMLV-II 55 30.362 0.129 I61K/Q68R/Q79R/L99R/E282D MMLV-II 55 31.473 0.070 I61M/Q68R/Q79R/L99R/E282D MMLV-II 55 32.892 0.286 Q68I/Q79H/L99K/E282M MMLV-II 55 33.872 0.131 I61M/Q68I/Q79H/L99K/E282M

TABLE 16 Two-Step cDNA synthesis by MMLV RT triple and more mutants by random hexamer priming. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature of Ct Ct Standard MMLV RT Variant Reaction (° C.) Mean Deviation MMLV-II 42 24.675 0.054 MMLV-II L99R/E282D 42 24.864 0.043 MMLV-II Q68R/L99R 42 24.577 0.066 MMLV-II Q79R/L99R 42 24.630 0.103 MMLV-II Q68R/Q79R 42 24.496 0.050 MMLV-II Q68R/L99R/E282D 42 24.549 0.059 MMLV-II Q79R/L99R/E282D 42 24.625 0.013 MMLV-II Q68R/Q79R/E282D 42 24.623 0.083 MMLV-II Q68R/Q79R/L99R 42 24.494 0.070 MMLV-II 42 24.422 0.035 Q68R/Q79R/L99R/E282D MMLV-II 42 24.517 0.066 Q68R/Q79R/L99K/E282D MMLV-II 42 24.324 0.059 Q68R/Q79R/L99N/E282D MMLV-II 42 24.488 0.070 Q68I/Q79R/L99R/E282D MMLV-II 42 24.501 0.041 Q68K/Q79R/L99R/E282D MMLV-II 42 26.574 0.029 Q68R/Q79H/L99R/E282D MMLV-II 42 24.496 0.055 Q68R/Q79I/L99R/E282D MMLV-II 42 24.382 0.043 Q68R/Q79R/L99R/E282M MMLV-II 42 24.617 0.109 Q68R/Q79R/L99R/E282W MMLV-II 42 24.391 0.045 I61K/Q68R/Q79R/L99R/E282D MMLV-II 42 24.426 0.028 I61M/Q68R/Q79R/L99R/E282D MMLV-II 42 24.660 0.027 Q68I/Q79H/L99K/E282M MMLV-II 42 24.949 0.052 I61M/Q68I/Q79H/L99K/E282M MMLV-II 55 32.082 0.095 MMLV-II L99R/E282D 55 31.612 0.190 MMLV-II Q68R/L99R 55 30.349 0.041 MMLV-II Q79R/L99R 55 30.494 0.094 MMLV-II Q68R/Q79R 55 29.735 0.153 MMLV-II Q68R/L99R/E282D 55 30.724 0.045 MMLV-II Q79R/L99R/E282D 55 30.774 0.152 MMLV-II Q68R/Q79R/E282D 55 30.232 0.079 MMLV-II Q68R/Q79R/L99R 55 28.270 0.340 MMLV-II 55 26.673 0.143 Q68R/Q79R/L99R/E282D MMLV-II 55 28.258 0.018 Q68R/Q79R/L99K/E282D MMLV-II 55 28.973 0.116 Q68R/Q79R/L99N/E282D MMLV-II 55 31.617 0.071 Q68I/Q79R/L99R/E282D MMLV-II 55 28.994 0.110 Q68K/Q79R/L99R/E282D MMLV-II 55 35.664 0.695 Q68R/Q79H/L99R/E282D MMLV-II 55 30.265 0.116 Q68R/Q79I/L99R/E282D MMLV-II 55 29.765 0.059 Q68R/Q79R/L99R/E282M MMLV-II 55 30.535 0.424 Q68R/Q79R/L99R/E282W MMLV-II 55 28.878 0.038 I61K/Q68R/Q79R/L99R/E282D MMLV-II 55 29.778 0.081 I61M/Q68R/Q79R/L99R/E282D MMLV-II 55 31.836 0.222 Q68I/Q79H/L99K/E282M MMLV-II 55 31.984 0.223 I61M/Q68I/Q79H/L99K/E282M f. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA Over a Wide Range of Temperatures

MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Oligo-dT or random hexamer priming conditions and reaction temperatures were adjusted for the two-step reactions and RTase concentration was normalized to 31 nM. The two-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see Tables 17 and 18).

Six of the nine MMLV RTase triple or more mutant variants were found to exhibit high overall activity as compared to the other MMLV RTase stacked mutant variants over a wide range of temperatures, spanning from 37.0 to 65° C., regardless of which priming method used. All of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The six MMLV RTase mutant variants that were found to exhibit the highest overall activity at a wide range of temperatures were Q68R/Q79R/L99R, Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99K/E282D, Q68R/Q79R/L99N/E282D, 161K/Q68R/Q79R/L99R/E282D and 161 M/Q68R/Q79R/L99R/E282D.

TABLE 17 Two-Step cDNA synthesis by MMLV RT triple and more mutants by Oligo-dT priming. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature of Ct Ct Standard MMLV RT Variant Reaction (° C.) Mean Deviation MMLV-II 37.0 26.593 0.020 MMLV-II Q79R/L99R/E282D 37.0 25.713 0.024 MMLV-II Q68R/Q79R/L99R 37.0 25.164 0.059 MMLV-II 37.0 25.163 0.035 Q68R/Q79R/L99R/E282D MMLV-II 37.0 25.135 0.078 Q68R/Q79R/L99K/E282D MMLV-II 37.0 25.693 0.048 Q68R/Q79R/L99N/E282D MMLV-II 37.0 25.491 0.062 Q68K/Q79R/L99R/E282D MMLV-II 37.0 25.450 0.083 Q68R/Q79R/L99R/E282M MMLV-II 37.0 25.094 0.071 I61K/Q68R/Q79R/L99R/E282D MMLV-II 37.0 25.356 0.034 I61M/Q68R/Q79R/L99R/E282D MMLV-II 37.8 26.623 0.062 MMLV-II Q79R/L99R/E282D 37.8 25.516 0.078 MMLV-II Q68R/Q79R/L99R 37.8 25.251 0.094 MMLV-II 37.8 24.987 0.050 Q68R/Q79R/L99R/E282D MMLV-II 37.8 25.093 0.084 Q68R/Q79R/L99K/E282D MMLV-II 37.8 25.273 0.095 Q68R/Q79R/L99N/E282D MMLV-II 37.8 25.310 0.079 Q68K/Q79R/L99R/E282D MMLV-II 37.8 25.545 0.044 Q68R/Q79R/L99R/E282M MMLV-II 37.8 25.144 0.196 I61K/Q68R/Q79R/L99R/E282D MMLV-II 37.8 25.302 0.035 I61M/Q68R/Q79R/L99R/E282D MMLV-II 39.5 26.430 0.074 MMLV-II Q79R/L99R/E282D 39.5 25.067 0.026 MMLV-II Q68R/Q79R/L99R 39.5 25.138 0.050 MMLV-II 39.5 24.788 0.022 Q68R/Q79R/L99R/E282D MMLV-II 39.5 24.842 0.071 Q68R/Q79R/L99K/E282D MMLV-II 39.5 24.892 0.042 Q68R/Q79R/L99N/E282D MMLV-II 39.5 25.047 0.038 Q68K/Q79R/L99R/E282D MMLV-II 39.5 25.249 0.081 Q68R/Q79R/L99R/E282M MMLV-II 39.5 24.845 0.130 I61K/Q68R/Q79R/L99R/E282D MMLV-II 39.5 25.130 0.072 I61M/Q68R/Q79R/L99R/E282D MMLV-II 42.0 25.485 0.052 MMLV-II Q79R/L99R/E282D 42.0 24.941 0.024 MMLV-II Q68R/Q79R/L99R 42.0 24.848 0.101 MMLV-II 42.0 24.802 0.009 Q68R/Q79R/L99R/E282D MMLV-II 42.0 24.805 0.008 Q68R/Q79R/L99K/E282D MMLV-II 42.0 24.744 0.076 Q68R/Q79R/L99N/E282D MMLV-II 42.0 24.893 0.073 Q68K/Q79R/L99R/E282D MMLV-II 42.0 24.968 0.031 Q68R/Q79R/L99R/E282M MMLV-II 42.0 24.933 0.088 I61K/Q68R/Q79R/L99R/E282D MMLV-II 42.0 24.821 0.045 I61M/Q68R/Q79R/L99R/E282D MMLV-II 45.2 25.776 0.028 MMLV-II Q79R/L99R/E282D 45.2 24.902 0.034 MMLV-II Q68R/Q79R/L99R 45.2 24.792 0.055 MMLV-II 45.2 24.705 0.092 Q68R/Q79R/L99R/E282D MMLV-II 45.2 24.791 0.009 Q68R/Q79R/L99K/E282D MMLV-II 45.2 24.890 0.071 Q68R/Q79R/L99N/E282D MMLV-II 45.2 25.420 0.101 Q68K/Q79R/L99R/E282D MMLV-II 45.2 25.196 0.086 Q68R/Q79R/L99R/E282M MMLV-II 45.2 24.823 0.079 I61K/Q68R/Q79R/L99R/E282D MMLV-II 45.2 24.720 0.006 I61M/Q68R/Q79R/L99R/E282D MMLV-II 47.8 27.932 0.049 MMLV-II Q79R/L99R/E282D 47.8 24.858 0.063 MMLV-II Q68R/Q79R/L99R 47.8 24.685 0.095 MMLV-II 47.8 24.689 0.067 Q68R/Q79R/L99R/E282D MMLV-II 47.8 24.620 0.072 Q68R/Q79R/L99K/E282D MMLV-II 47.8 24.780 0.039 Q68R/Q79R/L99N/E282D MMLV-II 47.8 24.855 0.018 Q68K/Q79R/L99R/E282D MMLV-II 47.8 24.961 0.040 Q68R/Q79R/L99R/E282M MMLV-II 47.8 24.681 0.076 I61K/Q68R/Q79R/L99R/E282D MMLV-II 47.8 24.759 0.055 I61M/Q68R/Q79R/L99R/E282D MMLV-II 49.2 30.393 0.118 MMLV-II Q79R/L99R/E282D 49.2 24.974 0.090 MMLV-II Q68R/Q79R/L99R 49.2 24.794 0.056 MMLV-II 49.2 24.720 0.100 Q68R/Q79R/L99R/E282D MMLV-II 49.2 25.007 0.096 Q68R/Q79R/L99K/E282D MMLV-II 49.2 25.304 0.147 Q68R/Q79R/L99N/E282D MMLV-II 49.2 25.273 0.066 Q68K/Q79R/L99R/E282D MMLV-II 49.2 25.560 0.019 Q68R/Q79R/L99R/E282M MMLV-II 49.2 24.719 0.177 I61K/Q68R/Q79R/L99R/E282D MMLV-II 49.2 25.123 0.034 I61M/Q68R/Q79R/L99R/E282D MMLV-II 50.0 30.870 0.210 MMLV-II Q79R/L99R/E282D 50.0 26.677 0.090 MMLV-II Q68R/Q79R/L99R 50.0 25.381 0.049 MMLV-II 50.0 24.820 0.064 Q68R/Q79R/L99R/E282D MMLV-II 50.0 25.348 0.098 Q68R/Q79R/L99K/E282D MMLV-II 50.0 25.287 0.064 Q68R/Q79R/L99N/E282D MMLV-II 50.0 25.208 0.085 Q68K/Q79R/L99R/E282D MMLV-II 50.0 25.790 0.051 Q68R/Q79R/L99R/E282M MMLV-II 50.0 24.840 0.071 I61K/Q68R/Q79R/L99R/E282D MMLV-II 50.0 25.317 0.042 I61M/Q68R/Q79R/L99R/E282D MMLV-II 51.0 27.914 0.002 MMLV-II Q79R/L99R/E282D 51.0 25.561 0.069 MMLV-II Q68R/Q79R/L99R 51.0 25.225 0.069 MMLV-II 51.0 24.726 0.034 Q68R/Q79R/L99R/E282D MMLV-II 51.0 25.324 0.071 Q68R/Q79R/L99K/E282D MMLV-II 51.0 25.157 0.062 Q68R/Q79R/L99N/E282D MMLV-II 51.0 25.275 0.039 Q68K/Q79R/L99R/E282D MMLV-II 51.0 25.938 0.095 Q68R/Q79R/L99R/E282M MMLV-II 51.0 25.821 0.072 I61K/Q68R/Q79R/L99R/E282D MMLV-II 51.0 25.053 0.044 I61M/Q68R/Q79R/L99R/E282D MMLV-II 51.9 28.602 0.059 MMLV-II Q79R/L99R/E282D 51.9 25.975 0.024 MMLV-II Q68R/Q79R/L99R 51.9 25.256 0.075 MMLV-II 51.9 24.903 0.050 Q68R/Q79R/L99R/E282D MMLV-II 51.9 25.163 0.169 Q68R/Q79R/L99K/E282D MMLV-II 51.9 25.272 0.011 Q68R/Q79R/L99N/E282D MMLV-II 51.9 25.491 0.075 Q68K/Q79R/L99R/E282D MMLV-II 51.9 25.878 0.038 Q68R/Q79R/L99R/E282M MMLV-II 51.9 26.071 0.044 I61K/Q68R/Q79R/L99R/E282D MMLV-II 51.9 25.419 0.067 I61M/Q68R/Q79R/L99R/E282D MMLV-II 53.8 26.412 0.082 MMLV-II Q79R/L99R/E282D 53.8 25.558 0.063 MMLV-II Q68R/Q79R/L99R 53.8 24.969 0.065 MMLV-II 53.8 25.356 0.063 Q68R/Q79R/L99R/E282D MMLV-II 53.8 25.460 0.056 Q68R/Q79R/L99K/E282D MMLV-II 53.8 25.769 0.118 Q68R/Q79R/L99N/E282D MMLV-II 53.8 26.251 0.103 Q68K/Q79R/L99R/E282D MMLV-II 53.8 26.310 0.174 Q68R/Q79R/L99R/E282M MMLV-II 53.8 25.701 0.106 I61K/Q68R/Q79R/L99R/E282D MMLV-II 53.8 26.412 0.082 I61M/Q68R/Q79R/L99R/E282D MMLV-II 56.5 29.343 0.085 MMLV-II Q79R/L99R/E282D 56.5 26.885 0.083 MMLV-II Q68R/Q79R/L99R 56.5 25.736 0.015 MMLV-II 56.5 25.223 0.016 Q68R/Q79R/L99R/E282D MMLV-II 56.5 25.900 0.039 Q68R/Q79R/L99K/E282D MMLV-II 56.5 25.930 0.031 Q68R/Q79R/L99N/E282D MMLV-II 56.5 25.869 0.204 Q68K/Q79R/L99R/E282D MMLV-II 56.5 26.622 0.067 Q68R/Q79R/L99R/E282M MMLV-II 56.5 25.817 0.089 I61K/Q68R/Q79R/L99R/E282D MMLV-II 56.5 26.290 0.009 I61M/Q68R/Q79R/L99R/E282D MMLV-II 59.9 29.693 0.047 MMLV-II Q79R/L99R/E282D 59.9 27.820 0.014 MMLV-II Q68R/Q79R/L99R 59.9 26.069 0.057 MMLV-II 59.9 25.374 0.061 Q68R/Q79R/L99R/E282D MMLV-II 59.9 26.066 0.053 Q68R/Q79R/L99K/E282D MMLV-II 59.9 25.873 0.018 Q68R/Q79R/L99N/E282D MMLV-II 59.9 26.278 0.073 Q68K/Q79R/L99R/E282D MMLV-II 59.9 27.068 0.075 Q68R/Q79R/L99R/E282M MMLV-II 59.9 26.863 0.025 I61K/Q68R/Q79R/L99R/E282D MMLV-II 59.9 26.176 0.072 I61M/Q68R/Q79R/L99R/E282D MMLV-II 62.6 29.731 0.092 MMLV-II Q79R/L99R/E282D 62.6 27.161 0.035 MMLV-II Q68R/Q79R/L99R 62.6 25.929 0.026 MMLV-II 62.6 25.303 0.074 Q68R/Q79R/L99R/E282D MMLV-II 62.6 25.907 0.003 Q68R/Q79R/L99K/E282D MMLV-II 62.6 26.145 0.053 Q68R/Q79R/L99N/E282D MMLV-II 62.6 26.181 0.056 Q68K/Q79R/L99R/E282D MMLV-II 62.6 27.134 0.015 Q68R/Q79R/L99R/E282M MMLV-II 62.6 26.025 0.178 I61K/Q68R/Q79R/L99R/E282D MMLV-II 62.6 26.304 0.041 I61M/Q68R/Q79R/L99R/E282D MMLV-II 64.2 26.809 0.080 MMLV-II Q79R/L99R/E282D 64.2 27.325 0.038 MMLV-II Q68R/Q79R/L99R 64.2 26.131 0.018 MMLV-II 64.2 25.542 0.135 Q68R/Q79R/L99R/E282D MMLV-II 64.2 26.408 0.093 Q68R/Q79R/L99K/E282D MMLV-II 64.2 26.734 0.040 Q68R/Q79R/L99N/E282D MMLV-II 64.2 30.589 0.128 Q68K/Q79R/L99R/E282D MMLV-II 64.2 26.262 0.090 Q68R/Q79R/L99R/E282M MMLV-II 64.2 27.594 0.118 I61K/Q68R/Q79R/L99R/E282D MMLV-II 64.2 27.062 0.051 I61M/Q68R/Q79R/L99R/E282D MMLV-II 65.0 30.277 0.050 MMLV-II Q79R/L99R/E282D 65.0 27.119 0.065 MMLV-II Q68R/Q79R/L99R 65.0 26.078 0.025 MMLV-II 65.0 25.583 0.068 Q68R/Q79R/L99R/E282D MMLV-II 65.0 25.906 0.080 Q68R/Q79R/L99K/E282D MMLV-II 65.0 26.943 0.058 Q68R/Q79R/L99N/E282D MMLV-II 65.0 26.413 0.067 Q68K/Q79R/L99R/E282D MMLV-II 65.0 28.233 0.075 Q68R/Q79R/L99R/E282M MMLV-II 65.0 25.778 0.129 I61K/Q68R/Q79R/L99R/E282D MMLV-II 65.0 27.345 0.015 I61M/Q68R/Q79R/L99R/E282D

TABLE 18 Two-Step cDNA synthesis by MMLV RT triple and more mutants by random hexamer priming. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature of Ct Ct Standard MMLV RT Variant Reaction (° C.) Mean Deviation MMLV-II 37.0 25.827 0.120 MMLV-II Q79R/L99R/E282D 37.0 25.616 0.094 MMLV-II Q68R/Q79R/L99R 37.0 24.747 0.041 MMLV-II 37.0 24.595 0.034 Q68R/Q79R/L99R/E282D MMLV-II 37.0 24.917 0.078 Q68R/Q79R/L99K/E282D MMLV-II 37.0 24.817 0.024 Q68R/Q79R/L99N/E282D MMLV-II 37.0 24.757 0.032 Q68K/Q79R/L99R/E282D MMLV-II 37.0 24.754 0.062 Q68R/Q79R/L99R/E282M MMLV-II 37.0 24.883 0.106 I61K/Q68R/Q79R/L99R/E282D MMLV-II 37.0 24.776 0.028 I61M/Q68R/Q79R/L99R/E282D MMLV-II 37.8 25.609 0.038 MMLV-II Q79R/L99R/E282D 37.8 25.300 0.061 MMLV-II Q68R/Q79R/L99R 37.8 24.822 0.037 MMLV-II 37.8 24.690 0.044 Q68R/Q79R/L99R/E282D MMLV-II 37.8 24.884 0.033 Q68R/Q79R/L99K/E282D MMLV-II 37.8 24.665 0.022 Q68R/Q79R/L99N/E282D MMLV-II 37.8 24.846 0.021 Q68K/Q79R/L99R/E282D MMLV-II 37.8 24.882 0.043 Q68R/Q79R/L99R/E282M MMLV-II 37.8 24.846 0.059 I61K/Q68R/Q79R/L99R/E282D MMLV-II 37.8 24.723 0.023 I61M/Q68R/Q79R/L99R/E282D MMLV-II 39.5 25.455 0.020 MMLV-II Q79R/L99R/E282D 39.5 24.790 0.109 MMLV-II Q68R/Q79R/L99R 39.5 24.712 0.050 MMLV-II 39.5 24.543 0.005 Q68R/Q79R/L99R/E282D MMLV-II 39.5 24.714 0.035 Q68R/Q79R/L99K/E282D MMLV-II 39.5 24.520 0.084 Q68R/Q79R/L99N/E282D MMLV-II 39.5 24.752 0.047 Q68K/Q79R/L99R/E282D MMLV-II 39.5 24.850 0.054 Q68R/Q79R/L99R/E282M MMLV-II 39.5 24.698 0.059 I61K/Q68R/Q79R/L99R/E282D MMLV-II 39.5 24.682 0.024 I61M/Q68R/Q79R/L99R/E282D MMLV-II 42.0 25.136 0.034 MMLV-II Q79R/L99R/E282D 42.0 24.760 0.052 MMLV-II Q68R/Q79R/L99R 42.0 24.637 0.037 MMLV-II 42.0 24.449 0.008 Q68R/Q79R/L99R/E282D MMLV-II 42.0 24.650 0.068 Q68R/Q79R/L99K/E282D MMLV-II 42.0 24.477 0.055 Q68R/Q79R/L99N/E282D MMLV-II 42.0 24.624 0.029 Q68K/Q79R/L99R/E282D MMLV-II 42.0 24.627 0.044 Q68R/Q79R/L99R/E282M MMLV-II 42.0 24.718 0.083 I61K/Q68R/Q79R/L99R/E282D MMLV-II 42.0 24.532 0.021 I61M/Q68R/Q79R/L99R/E282D MMLV-II 45.2 25.079 0.017 MMLV-II Q79R/L99R/E282D 45.2 24.624 0.026 MMLV-II Q68R/Q79R/L99R 45.2 24.525 0.021 MMLV-II 45.2 24.430 0.014 Q68R/Q79R/L99R/E282D MMLV-II 45.2 24.525 0.037 Q68R/Q79R/L99K/E282D MMLV-II 45.2 34.853 0.705 Q68R/Q79R/L99N/E282D MMLV-II 45.2 24.653 0.055 Q68K/Q79R/L99R/E282D MMLV-II 45.2 24.552 0.060 Q68R/Q79R/L99R/E282M MMLV-II 45.2 24.595 0.027 I61K/Q68R/Q79R/L99R/E282D MMLV-II 45.2 24.493 0.016 I61M/Q68R/Q79R/L99R/E282D MMLV-II 47.8 25.346 0.007 MMLV-II Q79R/L99R/E282D 47.8 24.521 0.097 MMLV-II Q68R/Q79R/L99R 47.8 24.605 0.018 MMLV-II 47.8 24.333 0.107 Q68R/Q79R/L99R/E282D MMLV-II 47.8 24.516 0.043 Q68R/Q79R/L99K/E282D MMLV-II 47.8 24.527 0.026 Q68R/Q79R/L99N/E282D MMLV-II 47.8 24.539 0.064 Q68K/Q79R/L99R/E282D MMLV-II 47.8 24.631 0.019 Q68R/Q79R/L99R/E282M MMLV-II 47.8 24.227 0.260 I61K/Q68R/Q79R/L99R/E282D MMLV-II 47.8 24.441 0.030 I61M/Q68R/Q79R/L99R/E282D MMLV-II 49.2 25.791 0.064 MMLV-II Q79R/L99R/E282D 49.2 24.700 0.033 MMLV-II Q68R/Q79R/L99R 49.2 24.658 0.008 MMLV-II 49.2 24.471 0.069 Q68R/Q79R/L99R/E282D MMLV-II 49.2 24.590 0.024 Q68R/Q79R/L99K/E282D MMLV-II 49.2 24.482 0.099 Q68R/Q79R/L99N/E282D MMLV-II 49.2 24.549 0.028 Q68K/Q79R/L99R/E282D MMLV-II 49.2 24.753 0.030 Q68R/Q79R/L99R/E282M MMLV-II 49.2 24.499 0.157 I61K/Q68R/Q79R/L99R/E282D MMLV-II 49.2 24.559 0.033 I61M/Q68R/Q79R/L99R/E282D MMLV-II 50.0 26.267 0.025 MMLV-II Q79R/L99R/E282D 50.0 24.729 0.047 MMLV-II Q68R/Q79R/L99R 50.0 24.462 0.040 MMLV-II 50.0 24.412 0.035 Q68R/Q79R/L99R/E282D MMLV-II 50.0 24.438 0.090 Q68R/Q79R/L99K/E282D MMLV-II 50.0 24.509 0.050 Q68R/Q79R/L99N/E282D MMLV-II 50.0 24.405 0.059 Q68K/Q79R/L99R/E282D MMLV-II 50.0 24.547 0.041 Q68R/Q79R/L99R/E282M MMLV-II 50.0 24.504 0.005 I61K/Q68R/Q79R/L99R/E282D MMLV-II 50.0 24.481 0.009 I61M/Q68R/Q79R/L99R/E282D MMLV-II 51.0 27.277 0.058 MMLV-II Q79R/L99R/E282D 51.0 25.694 0.104 MMLV-II Q68R/Q79R/L99R 51.0 24.579 0.037 MMLV-II 51.0 24.364 0.019 Q68R/Q79R/L99R/E282D MMLV-II 51.0 24.849 0.041 Q68R/Q79R/L99K/E282D MMLV-II 51.0 24.899 0.121 Q68R/Q79R/L99N/E282D MMLV-II 51.0 24.980 0.048 Q68K/Q79R/L99R/E282D MMLV-II 51.0 25.292 0.065 Q68R/Q79R/L99R/E282M MMLV-II 51.0 25.147 0.100 I61K/Q68R/Q79R/L99R/E282D MMLV-II 51.0 25.034 0.075 I61M/Q68R/Q79R/L99R/E282D MMLV-II 51.9 28.797 0.055 MMLV-II Q79R/L99R/E282D 51.9 26.585 0.011 MMLV-II Q68R/Q79R/L99R 51.9 25.021 0.036 MMLV-II 51.9 24.763 0.028 Q68R/Q79R/L99R/E282D MMLV-II 51.9 25.392 0.012 Q68R/Q79R/L99K/E282D MMLV-II 51.9 25.543 0.087 Q68R/Q79R/L99N/E282D MMLV-II 51.9 25.549 0.058 Q68K/Q79R/L99R/E282D MMLV-II 51.9 26.025 0.065 Q68R/Q79R/L99R/E282M MMLV-II 51.9 26.087 0.024 I61K/Q68R/Q79R/L99R/E282D MMLV-II 51.9 25.756 0.054 I61M/Q68R/Q79R/L99R/E282D MMLV-II 53.8 30.985 0.073 MMLV-II Q79R/L99R/E282D 53.8 29.356 0.044 MMLV-II Q68R/Q79R/L99R 53.8 26.370 0.041 MMLV-II 53.8 25.580 0.049 Q68R/Q79R/L99R/E282D MMLV-II 53.8 26.682 0.029 Q68R/Q79R/L99K/E282D MMLV-II 53.8 26.438 0.031 Q68R/Q79R/L99N/E282D MMLV-II 53.8 27.024 0.042 Q68K/Q79R/L99R/E282D MMLV-II 53.8 28.314 0.051 Q68R/Q79R/L99R/E282M MMLV-II 53.8 27.489 0.025 I61K/Q68R/Q79R/L99R/E282D MMLV-II 53.8 27.871 0.118 I61M/Q68R/Q79R/L99R/E282D MMLV-II 56.5 33.313 0.164 MMLV-II Q79R/L99R/E282D 56.5 32.626 0.113 MMLV-II Q68R/Q79R/L99R 56.5 30.047 0.089 MMLV-II 56.5 29.183 0.155 Q68R/Q79R/L99R/E282D MMLV-II 56.5 30.750 0.051 Q68R/Q79R/L99K/E282D MMLV-II 56.5 30.403 0.095 Q68R/Q79R/L99N/E282D MMLV-II 56.5 31.707 0.111 Q68K/Q79R/L99R/E282D MMLV-II 56.5 31.878 0.093 Q68R/Q79R/L99R/E282M MMLV-II 56.5 32.235 0.291 I61K/Q68R/Q79R/L99R/E282D MMLV-II 56.5 32.395 0.105 I61M/Q68R/Q79R/L99R/E282D MMLV-II 59.9 34.408 0.498 MMLV-II Q79R/L99R/E282D 59.9 36.798 2.131 MMLV-II Q68R/Q79R/L99R 59.9 33.997 0.035 MMLV-II 59.9 32.009 0.051 Q68R/Q79R/L99R/E282D MMLV-II 59.9 33.685 0.317 Q68R/Q79R/L99K/E282D MMLV-II 59.9 33.083 0.163 Q68R/Q79R/L99N/E282D MMLV-II 59.9 34.160 0.066 Q68K/Q79R/L99R/E282D MMLV-II 59.9 33.650 0.161 Q68R/Q79R/L99R/E282M MMLV-II 59.9 33.341 0.096 I61K/Q68R/Q79R/L99R/E282D MMLV-II 59.9 34.439 0.222 I61M/Q68R/Q79R/L99R/E282D MMLV-II 62.6 35.163 0.447 MMLV-II Q79R/L99R/E282D 62.6 37.138 1.603 MMLV-II Q68R/Q79R/L99R 62.6 34.108 0.604 MMLV-II 62.6 32.539 0.060 Q68R/Q79R/L99R/E282D MMLV-II 62.6 34.175 0.421 Q68R/Q79R/L99K/E282D MMLV-II 62.6 33.726 0.622 Q68R/Q79R/L99N/E282D MMLV-II 62.6 34.376 0.408 Q68K/Q79R/L99R/E282D MMLV-II 62.6 33.792 0.231 Q68R/Q79R/L99R/E282M MMLV-II 62.6 33.768 0.387 I61K/Q68R/Q79R/L99R/E282D MMLV-II 62.6 34.428 0.085 I61M/Q68R/Q79R/L99R/E282D MMLV-II 64.2 37.284 0.764 MMLV-II Q79R/L99R/E282D 64.2 36.661 0.192 MMLV-II Q68R/Q79R/L99R 64.2 34.463 0.213 MMLV-II 64.2 32.992 0.023 Q68R/Q79R/L99R/E282D MMLV-II 64.2 34.805 0.472 Q68R/Q79R/L99K/E282D MMLV-II 64.2 34.060 0.043 Q68R/Q79R/L99N/E282D MMLV-II 64.2 34.508 0.302 Q68K/Q79R/L99R/E282D MMLV-II 64.2 34.481 0.078 Q68R/Q79R/L99R/E282M MMLV-II 64.2 34.231 0.253 I61K/Q68R/Q79R/L99R/E282D MMLV-II 64.2 35.049 0.885 I61M/Q68R/Q79R/L99R/E282D MMLV-II 65.0 35.809 0.511 MMLV-II Q79R/L99R/E282D 65.0 35.932 0.372 MMLV-II Q68R/Q79R/L99R 65.0 34.979 0.856 MMLV-II 65.0 33.293 0.319 Q68R/Q79R/L99R/E282D MMLV-II 65.0 34.974 0.536 Q68R/Q79R/L99K/E282D MMLV-II 65.0 34.862 0.268 Q68R/Q79R/L99N/E282D MMLV-II 65.0 34.363 0.201 Q68K/Q79R/L99R/E282D MMLV-II 65.0 34.687 0.666 Q68R/Q79R/L99R/E282M MMLV-II 65.0 34.246 0.563 I61K/Q68R/Q79R/L99R/E282D MMLV-II 65.0 34.872 0.467 I61M/Q68R/Q79R/L99R/E282D

Example 6: Reverse Transcriptase Mutant Evaluation by Oligo dT or Random Priming

This example demonstrates the procedure used to evaluate each mutant RTase's ability to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) compared to the base construct of MMLV RTase. The mutant MMLV RTases were tested by two priming conditions: Oligo dT only and random hexamer priming using a standard two-step cDNA synthesis as described in Example 5.

The reactions were analyzed and reported by Ct value (Tables 19 and 20). Four mutant variants of MMLV RTase showed an increase in the overall activity using oligo dT priming compared to the base construct, Q299E, T332E and V433R. Eight mutant variants of MMLV RTase showed an increase in the overall activity using random priming compared to the base construct, P76R, L82R, I125R, Y271A, L280A, L280R, T328R and V433R.

TABLE 19 Two-Step cDNA Synthesis by MMLV-RT single mutants using oligo dT priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 40.000 0.000 MMLV-II D209A 40.000 0.000 MMLV-II D209E 40.000 0.000 MMLV-II D209R 40.000 0.000 MMLV-II D83 A 40.000 0.000 MMLV-II D83E 40.000 0.000 MMLV-II D83R 40.000 0.000 MMLV-II E201A 40.000 0.000 MMLV-II E201D 40.000 0.000 MMLV-II E201R 40.000 0.000 MMLV-II E367A 40.000 0.000 MMLV-II E367D 40.000 0.000 MMLV-II E367R 40.000 0.000 MMLV-II E596A 40.000 0.000 MMLV-II E596D 40.000 0.000 MMLV-II E596R 40.000 0.000 MMLV-II F210A 40.000 0.000 MMLV-II F210E 40.000 0.000 MMLV-II F210R 40.000 0.000 MMLV-II F369A 40.000 0.000 MMLV-II F369E 40.000 0.000 MMLV-II F369R 40.000 0.000 MMLV-II G308A 40.000 0.000 MMLV-II G308E 40.000 0.000 MMLV-II G308R 40.000 0.000 MMLV-II G331A 40.000 0.000 MMLV-II G331E 40.000 0.000 MMLV-II G331R 40.000 0.000 MMLV-II G73A 40.000 0.000 MMLV-II G73E 40.000 0.000 MMLV-II G73R 40.000 0.000 MMLV-II H77A 40.000 0.000 MMLV-II H77E 40.000 0.000 MMLV-II H77R 40.000 0.000 MMLV-II I125A 40.000 0.000 MMLV-II I125E 40.000 0.000 MMLV-II I125R 40.000 0.000 MMLV-II I212A 40.000 0.000 MMLV-II I212E 40.000 0.000 MMLV-II I212R 40.000 0.000 MMLV-II I593A 40.000 0.000 MMLV-II I593E 40.000 0.000 MMLV-II I593R 40.000 0.000 MMLV-II I597A 40.000 0.000 MMLV-II I597E 40.000 0.000 MMLV-II I597R 40.000 0.000 MMLV-II K285A 40.000 0.000 MMLV-II K285E 40.000 0.000 MMLV-II K285R 40.000 0.000 MMLV-II K348A 40.000 0.000 MMLV-II K348E 40.000 0.000 MMLV-II K348R 40.000 0.000 MMLV-II L198A 40.000 0.000 MMLV-II L198E 40.000 0.000 MMLV-II L198R 40.000 0.000 MMLV-II L280A 40.000 0.000 MMLV-II L280E 40.000 0.000 MMLV-II L280R 40.000 0.000 MMLV-II L352A 40.000 0.000 MMLV-II L352E 40.000 0.000 MMLV-II L352R 40.000 0.000 MMLV-II L357A 40.000 0.000 MMLV-II L357E 40.000 0.000 MMLV-II L357R 40.000 0.000 MMLV-II L82A 40.000 0.000 MMLV-II L82E 40.000 0.000 MMLV-II L82R 40.000 0.000 MMLV-II N335A 39.787 0.302 MMLV-II N335E 40.000 0.000 MMLV-II N335R 40.000 0.000 MMLV-II P76A 40.000 0.000 MMLV-II P76E 40.000 0.000 MMLV-II P76R 40.000 0.000 MMLV-II Q213A 40.000 0.000 MMLV-II Q213E 40.000 0.000 MMLV-II Q213R 40.000 0.000 MMLV-II Q299A 40.000 0.000 MMLV-II Q299E 37.177 3.993 MMLV-II Q299R 40.000 0.000 MMLV-II Q654A 40.000 0.000 MMLV-II Q654E 40.000 0.000 MMLV-II Q654R 40.000 0.000 MMLV-II R205A 40.000 0.000 MMLV-II R205E 39.947 0.075 MMLV-II R205K 40.000 0.000 MMLV-II R211A 40.000 0.000 MMLV-II R211E 40.000 0.000 MMLV-II R211K 40.000 0.000 MMLV-II R311A 40.000 0.000 MMLV-II R311E 40.000 0.000 MMLV-II R311K 40.000 0.000 MMLV-II R389A 40.000 0.000 MMLV-II R389E 40.000 0.000 MMLV-II R389K 40.000 0.000 MMLV-II R650A 40.000 0.000 MMLV-II R650E 40.000 0.000 MMLV-II R650K 40.000 0.000 MMLV-II R657A 40.000 0.000 MMLV-II R657E 39.965 0.050 MMLV-II R657K 40.000 0.000 MMLV-II S67A 40.000 0.000 MMLV-II S67E 40.000 0.000 MMLV-II S67R 36.816 0.703 MMLV-II T328A 40.000 0.000 MMLV-II T328E 40.000 0.000 MMLV-II T328R 40.000 0.000 MMLV-II T332A 39.750 0.354 MMLV-II T332E 38.461 2.177 MMLV-II T332R 40.000 0.000 MMLV-II V129A 40.000 0.000 MMLV-II V129E 40.000 0.000 MMLV-II V129R 40.000 0.000 MMLV-II V433A 40.000 0.000 MMLV-II V433E 40.000 0.000 MMLV-II V433R 38.884 0.806 MMLV-II V476A 40.000 0.000 MMLV-II V476E 40.000 0.000 MMLV-II V476R 40.000 0.000 MMLV-II Y271A 40.000 0.000 MMLV-II Y271E 40.000 0.000 MMLV-II Y271R 40.000 0.000 MMLV-IV 31.467 0.190

TABLE 20 Two-Step cDNA Synthesis by MMLV-RT single mutants using random priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 40.000 0.000 MMLV-II D209A 40.000 0.000 MMLV-II D209E 40.000 0.000 MMLV-II D209R 40.000 0.000 MMLV-II D83A 40.000 0.000 MMLV-II D83E 40.000 0.000 MMLV-II D83R 40.000 0.000 MMLV-II E201A 40.000 0.000 MMLV-II E201D 40.000 0.000 MMLV-II E201R 40.000 0.000 MMLV-II E367A 40.000 0.000 MMLV-II E367D 40.000 0.000 MMLV-II E367R 40.000 0.000 MMLV-II E596A 40.000 0.000 MMLV-II E596D 40.000 0.000 MMLV-II E596R 40.000 0.000 MMLV-II F210A 40.000 0.000 MMLV-II F210E 40.000 0.000 MMLV-II F210R 40.000 0.000 MMLV-II F369A 40.000 0.000 MMLV-II F369E 40.000 0.000 MMLV-II F369R 40.000 0.000 MMLV-II G308A 40.000 0.000 MMLV-II G308E 40.000 0.000 MMLV-II G308R 40.000 0.000 MMLV-II G331A 40.000 0.000 MMLV-II G331E 40.000 0.000 MMLV-II G331R 40.000 0.000 MMLV-II G73A 40.000 0.000 MMLV-II G73E 40.000 0.000 MMLV-II G73R 40.000 0.000 MMLV-II H77A 39.708 0.412 MMLV-II H77E 40.000 0.000 MMLV-II H77R 40.000 0.000 MMLV-II I125A 40.000 0.000 MMLV-II I125E 40.000 0.000 MMLV-II I125R 39.449 0.779 MMLV-II I212A 40.000 0.000 MMLV-II I212E 40.000 0.000 MMLV-II I212R 40.000 0.000 MMLV-II I593A 40.000 0.000 MMLV-II I593E 40.000 0.000 MMLV-II I593R 40.000 0.000 MMLV-II I597A 40.000 0.000 MMLV-II I597E 40.000 0.000 MMLV-II I597R 40.000 0.000 MMLV-II K285A 40.000 0.000 MMLV-II K285E 40.000 0.000 MMLV-II K285R 39.783 0.308 MMLV-II K348A 40.000 0.000 MMLV-II K348E 40.000 0.000 MMLV-II K348R 40.000 0.000 MMLV-II L198A 40.000 0.000 MMLV-II L198E 40.000 0.000 MMLV-II L198R 40.000 0.000 MMLV-II L280A 39.503 0.703 MMLV-II L280E 40.000 0.000 MMLV-II L280R 38.762 1.751 MMLV-II L352A 39.778 0.313 MMLV-II L352E 40.000 0.000 MMLV-II L352R 40.000 0.000 MMLV-II L357A 40.000 0.000 MMLV-II L357E 40.000 0.000 MMLV-II L357R 40.000 0.000 MMLV-II L82A 40.000 0.000 MMLV-II L82E 39.673 0.462 MMLV-II L82R 38.926 1.518 MMLV-II N335A 39.876 0.175 MMLV-II N335E 40.000 0.000 MMLV-II N335R 39.861 0.196 MMLV-II P76A 40.000 0.000 MMLV-II P76E 40.000 0.000 MMLV-II P76R 39.535 0.658 MMLV-II Q213A 40.000 0.000 MMLV-II Q213E 40.000 0.000 MMLV-II Q213R 40.000 0.000 MMLV-II Q299A 40.000 0.000 MMLV-II Q299E 40.000 0.000 MMLV-II Q299R 40.000 0.000 MMLV-II Q654A 40.000 0.000 MMLV-II Q654E 40.000 0.000 MMLV-II Q654R 40.000 0.000 MMLV-II R205A 39.811 0.267 MMLV-II R205E 40.000 0.000 MMLV-II R205K 40.000 0.000 MMLV-II R211A 40.000 0.000 MMLV-II R211E 40.000 0.000 MMLV-II R211K 40.000 0.000 MMLV-II R311A 40.000 0.000 MMLV-II R311E 40.000 0.000 MMLV-II R311K 40.000 0.000 MMLV-II R389A 40.000 0.000 MMLV-II R389E 40.000 0.000 MMLV-II R389K 40.000 0.000 MMLV-II R650A 40.000 0.000 MMLV-II R650E 40.000 0.000 MMLV-II R650K 40.000 0.000 MMLV-II R657A 40.000 0.000 MMLV-II R657E 40.000 0.000 MMLV-II R657K 40.000 0.000 MMLV-II S67A 40.000 0.000 MMLV-II S67E 39.435 0.800 MMLV-II S67R 38.209 0.977 MMLV-II T328A 40.000 0.000 MMLV-II T328E 40.000 0.000 MMLV-II T328R 39.478 0.739 MMLV-II T332A 40.000 0.000 MMLV-II T332E 40.000 0.000 MMLV-II T332R 40.000 0.000 MMLV-II V129A 40.000 0.000 MMLV-II V129E 40.000 0.000 MMLV-II V129R 40.000 0.000 MMLV-II V433A 40.000 0.000 MMLV-II V433E 40.000 0.000 MMLV-II V433R 38.071 1.452 MMLV-II V476A 40.000 0.000 MMLV-II V476E 40.000 0.000 MMLV-II V476R 40.000 0.000 MMLV-II Y271A 39.466 0.755 MMLV-II Y271E 40.000 0.000 MMLV-II Y271R 40.000 0.000 MMLV-IV 31.850 0.183

In addition to the increased activity demonstrated in the MMLV RTase mutations Q299E, T332E, and V433R (Table 19), and the MMLV RTase mutations P76R, L82R, I125R, Y271A, L280A, L280R, T328R, and V433R (Table 20), further MMLV RTase mutations were selected by rational design and introduced by site-directed mutagenesis using standard PCR conditions and primers (Table 21).

TABLE 21 Sequences of primers used for cloning of MMLV RTase base construct and mutants into pET28b. All primers were ordered as DNA oligos from Integrated DNA Technologies. SEQ ID NO: Primer Name Primer Sequence (5′-3′) 700 MMLV V433R AGTTGACGATGGGTCAACCCTTACGTATCTTGGCTCCA SDM F CATGCTGTAGA 701 MMLV V433R TCTACAGCATGTGGAGCCAAGATACGTAAGGGTTGAC SDM R CCATCGTCAACT 702 MMLV I593E CGTTATGCTTTTGCAACAGCGCATGAGCATGGCGAAA SDM F TTTACCGCCGC 703 MMLV I593E GCGGCGGTAAATTTCGCCATGCTCATGCGCTGTTGCAA SDM R AAGCATAACG 704 MMLV Q299E TACGCCTAAGACGCCACGCGAGTTGCGTGAATTTTTG SDM F GGCACAGC 705 MMLV Q299E GCTGTGCCCAAAAATTCACGCAACTCGCGTGGCGTCTT SDM R AGGCGTA 706 MMLV L82Y GATTAAGCCACATATTCAGCGCTTGTATGACCAGGGG SDM F ATCTTGGTCC 707 MMLV L82Y GGACCAAGATCCCCTGGTCATACAAGCGCTGAATATG SDM R TGGCTTAATC 708 MMLV L280I TGCTGAAAGAAGGTCAACGTTGGATCACTGAAGCGCG SDM F TAAGGAGACC 709 MMLV L280I GGTCTCCTTACGCGCTTCAGTGATCCAACGTTGACCTT SDM R CTTTCAGCA 710 MMLV V433N AGTTGACGATGGGTCAACCCTTAAACATCTTGGCTCCA SDM F CATGCTGTAGA 711 MMLV V433N TCTACAGCATGTGGAGCCAAGATGTTTAAGGGTTGAC SDM R CCATCGTCAACT 712 MMLV I593W CGTTATGCTTTTGCAACAGCGCATTGGCATGGCGAAAT SDM F TTACCGCCGC 713 MMLV I593W GCGGCGGTAAATTTCGCCATGCCAATGCGCTGTTGCA SDM R AAAGCATAACG 714 MMLV T306K GCCAGTTGCGTGAATTTTTGGGCAAAGCGGGATTCTGT TOP CGTTTATGGATTCC 715 MMLV T306K GGAATCCATAAACGACAGAATCCCGCTTTGCCCAAAA BTM ATTCACGCAACTGGC

The resulting plasmids were transformed into E. coli BL21(DE3) cells for protein expression and proteins isolated through affinity and ion exchange chromatography (Table 22).

TABLE 22 Sequences of MMLV RTase base construct and mutant MMLV RTase constructs. SEQ ID NO: Construct Construct Sequence (DNA: 5′-3′ or AA) 716 MMLV-II RTase ATGACTTTAAATATTGAGGATGAGCATCGTTTA CATGAGACATCAAAAGAACCCGACGTGAGCTTA GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCAAGAGGCCCGCCTGGGGATTAAGCCA CATATTCAGCGCTTGCTGGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CTGCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGCTGACTGAAGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCCAGTTGCGTGAATTTTTGGGCACAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTAGTAATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATATC CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTTTAA 717 MMLV-II RTase MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP MSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHI HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 718 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L99R/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA E282D/Q299E/V433N/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG I593W TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGCTGGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGCTGACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCACAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTACGTATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATTGG CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 719 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L99R/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP E282D/Q299E/V433N/ MSREARLGIKPHIRRLLDQGILVPCQSPWNTPL I593W RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWLTDARKETVMGQPTPKI PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 720 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L99R/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA L2801/E282D/Q299E/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG V433N/I593W TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGCTGGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGATTACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCACAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTACGTATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC ACTCACGTTATGCTTTTGCAACAGCGCATTGG CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 721 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L99R/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP L280I/E282D/Q299E/ MSREARLGIKPHIRRLLDQGILVPCQSPWNTPL V433N/I593W RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 722 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG Q299E/V433N/I593W TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGTATGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGATTACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCACAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTACGTATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATTGG CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 723 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL Q299E/V433N/I593W RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 724 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG Q299E/T306K/V433N/ TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC I593W CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGTATGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGATTACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCAAAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTAAACATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATTGG CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 725 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL Q299E/T306K/V433N/ RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN I593W PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLNILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 726 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L99R/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA E282D/Q299E/T306K/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG V433R/I593E TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGCTGGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGCTGACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCAAAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTACGTATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATGAA CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 727 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L99R/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP E282D/Q299E/T306K/ MSREARLGIKPHIRRLLDQGILVPCQSPWNTPL V433R/I593E RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWLTDARKETVMGQPTPKI PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHE HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 728 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG Q299E/V433R/I593E TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGTATGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGATTACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCACAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTACGTATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATGAA CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 729 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL Q299E/V433R/I593E RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHE HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF 730 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG Q299E/T306K/V433R/ TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC I593E CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA ACGTCTACACCTGTCTCTATCAAACAGTACCCC ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA CATATTCGTCGCTTGTATGACCAGGGGATCTTG GTCCCATGTCAATCTCCGTGGAACACCCCCCTT CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA CGCGTAGAAGACATCCATCCGACTGTACCTAAT CCTTATAATCTGTTATCAGGCCTGCCCCCATCG CACCAATGGTATACAGTATTAGACTTGAAAGAC GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT GAAATGGGAATTTCGGGTCAGTTAACCTGGACT CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA TTGTTCGATGAAGCACTTCACCGTGACTTAGCA GACTTCCGTATCCAACACCCAGACTTAATTCTG TTACAGTATGTTGACGACCTTTTGTTGGCGGCA ACGTCTGAACTTGACTGTCAGCAAGGCACACGC GCGTTATTACAAACGTTAGGTAACTTAGGATAT CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA GAAGGTCAACGTTGGATTACTGATGCGCGTAAG GAGACCGTAATGGGGCAGCCTACGCCTAAGACG CCACGCGAATTGCGTGAATTTTTGGGCAAAGCG GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT GAAATGGCTGCACCCCTGTACCCCTTAACAAAA ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA CAGGGATATGCAAAAGGAGTATTAACCCAAAAG TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC GTTTTGACAAAGGATGCAGGTAAGTTGACGATG GGTCAACCCTTACGTATCTTGGCTCCACATGCT GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC TGGCTTTCTAATGCGCGCATGACCCACTATCAG GCGCTTCTGCTTGATACGGATCGTGTACAATTT GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT TGTTTAGATATTCTGGCCGAGGCACATGGGACG CGCCCTGATTTGACGGATCAGCCACTGCCTGAT GCCGACCATACATGGTATACTGGCGGCAGTAGT CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA GCCGTCACTACGGAGACCGAAGTTATCTGGGCC AAAGCGTTACCCGCGGGAACATCCGCGCAACGT GCACAGTTAATCGCTCTGACACAGGCCCTGAAG ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC AACTCACGTTATGCTTTTGCAACAGCGCATGAA CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA CCAAAACGCCTTTCGATCATTCATTGCCCGGGG CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG GCGATCACAGAGACCCCGGATACATCAACGCTG TTGATCGAAAACAGCTCTCCCTACACTAGCGAG CATTTT 731 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL Q299E/T306K/V433R/ RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN I593E PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTK TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHE HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPYTSEHF

For the standard two-step procedure, RTases (1 μL, 620 nM) were added to a reaction mixture containing RNA (20 ng), dNTPs (100 μM), oligo dT primer (5 ng/uL) or both random hexamers and oligo dT primers (5 ng/uL each), first strand synthesis buffer (1×, 50 mM potassium acetate, 20 mM tris-acetate, pH 7.9, 10 mM magnesium acetate, 0.6 M trehalose 100 μg/ml BSA, and 10 mM DTT), and SuperaseIN (0.17 U/μL) in a 20 μL volume. The reaction proceeded at 50 or 65° C. for 15 minutes, followed by 80° C. for 10 minutes.

The subsequent cDNA synthesized by the RTase mutants in this disclosure were quantified by qPCR amplification using an assay that identified the SFRS9 gene in human cells. The assay master mix was a composition of Integrated DNA Technologies PrimeTime© Gene Expression Master Mix (GEM, 1×), SFRS9 primer set (500 nM, Table 3) and SFRS9 probe (250 nM, Table 3). The assay master mix and synthesized cDNA were mixed at a 10:1 ratio for a final volume of 20 μL. The reaction proceeded on a qPCR (QuantStudio7 Flex) using the following method: 95° C. hold for 3 minutes, followed by 95° C. for 15 seconds and 60° C. for one minute for 40 cycles. The reactions were analyzed and reported by Ct value (Tables 23-25). All mutant variants of MMLV RTase showed an increase in the overall activity compared to the base construct and three mutant variants of MMLV RTase showed noteworthy activity compared to the others, Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/V433N/I593W; Q68R/Q79R/L99R/E282D/Q299E/T306K/V433R/I593E; and Q68R/Q79R/L83Y/L99R/L280I/E282D/Q299E/T306K/V433R/I593E.

TABLE 23 Two-Step cDNA Synthesis by MMLV-RT mutants using oligo dT priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. RT Temperature Ct Ct Standard MMLV-RT Variant (° C.) Mean Deviation MMLV-II 50 24.873 0.043 65 35.817 0.630 MMLV-II Q68R/Q79R/L99R/E282D 50 24.932 0.058 65 36.668 0.614 MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 50 24.750 0.036 65 35.782 1.366 MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433N/I593W 50 24.586 0.035 65 35.819 0.284 MMLV-II 50 24.638 0.028 Q68R/Q79R/L99R/E282D/L280EQ299E/V433N/I593W 65 34.319 0.343 MMLV-II 50 24.681 0.019 Q68R/Q79R/L82Y/L99R/E282D/L280I/Q299E/V433N/I593W 65 33.184 0.021

TABLE 24 Two-Step cDNA Synthesis by MMLV-RT mutants using oligo dT priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. RT Temperature Ct Ct Standard MMLV-RT Variant (° C.) Mean Deviation MMLV-II 50 24.887 0.041 65 32.730 0.053 MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 50 25.061 0.126 65 27.898 0.070 MMLV-II 50 24.849 0.101 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433N/I593W 65 26.607 0.077 MMLV-II 50 25.110 0.154 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/V433N/I593W 65 25.701 0.062 MMLV-II 50 24.990 0.088 Q68R/Q79R/L99R/E282D/Q299E/T306K/V433R/I593E 65 25.929 0.114 MMLV-II 50 25.133 0.114 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433R 65 27.032 0.141 I593E MMLV-II 50 24.817 0.122 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/ 65 25.721 0.187 V433R/I593E

TABLE 25 Two-Step cDNA Synthesis by MMLV-RT mutants using random priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. RT Temperature Ct Ct Standard MMLV-RT Variant (° C.) Mean Deviation MMLV-II 50 25.048 0.075 65 32.563 0.156 MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 50 25.002 0.027 65 28.062 0.106 MMLV-II 50 25.016 0.179 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433N/ 65 26.724 0.040 I593W MMLV-II 50 24.973 0.021 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/ 65 25.732 0.061 V433N/I593W MMLV-II 50 24.982 0.030 Q68R/Q79R/L99R/E282D/Q299E/T306K/V433R/I593E 65 26.006 0.020 MMLV-II 50 25.078 0.065 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433R/I593E 65 27.080 0.122 MMLV-II 50 25.074 0.094 Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/ 65 25.784 0.100 V433R/I593E

Example 7. Reverse Transcriptase Mutant Evaluation by Gene Specific Priming

This example demonstrates the procedure used to evaluate each mutant RTase's ability to synthesize cDNA from purified RNA ultramers (Integrated DNA Technologies) compared to the base construct of MMLV RTase. The mutant MMLV RTases were tested by a one-step addition of the RTase in GEM as described in Example 5. The reactions were analyzed and reported by Ct value (Table 26). Twelve mutant variants of MMLV RTase showed an increase in the overall activity compared to the base construct, H77A, D83E, D83R, Y271E, Q299E, G308E, F396A, V433R, I593E, I597A, and I597R.

TABLE 26 One-Step cDNA Synthesis by MMLV-RT single mutants by gene specific priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 29.065 0.277 MMLV-II D209A 29.583 0.166 MMLV-II D209E 28.900 0.088 MMLV-II D209R 29.266 0.068 MMLV-II D83 A 29.588 0.082 MMLV-II D83E 28.499 0.087 MMLV-II D83R 28.724 0.087 MMLV-II E201A 30.692 0.173 MMLV-II E201D 29.130 0.157 MMLV-II E201R 29.333 0.141 MMLV-II E367A 31.153 0.021 MMLV-II E367D 31.070 0.187 MMLV-II E367R 34.221 0.475 MMLV-II E596A 29.150 0.121 MMLV-II E596D 30.494 0.081 MMLV-II E596R 31.787 0.227 MMLV-II F210A 33.639 0.196 MMLV-II F210E 34.982 0.065 MMLV-II F210R 37.201 1.986 MMLV-II F369A 29.055 0.063 MMLV-II F369E 36.856 0.508 MMLV-II F369R 36.149 0.308 MMLV-II G308A 30.226 0.170 MMLV-II G308E 28.772 0.121 MMLV-II G308R 40.000 0.000 MMLV-II G331A 30.412 0.137 MMLV-II G331E 31.321 0.160 MMLV-II G331R 31.340 0.020 MMLV-II G73A 30.741 0.125 MMLV-II G73E 34.319 0.369 MMLV-II G73R 29.721 0.061 MMLV-II H77A 28.581 0.070 MMLV-II H77E 29.475 0.107 MMLV-II H77R 29.726 0.120 MMLV-II I125A 29.812 0.043 MMLV-II I125E 30.712 0.147 MMLV-II I125R 30.324 0.012 MMLV-II I212A 29.586 0.086 MMLV-II I212E 29.459 0.073 MMLV-II I212R 29.037 0.092 MMLV-II I593A 30.560 0.101 MMLV-II I593E 27.779 0.056 MMLV-II I593R 29.268 0.012 MMLV-II I597A 28.983 0.024 MMLV-II I597E 29.583 0.143 MMLV-II I597R 28.671 0.103 MMLV-II K285A 32.375 0.158 MMLV-II K285E 37.065 0.044 MMLV-II K285R 30.564 0.075 MMLV-II K348A 34.241 0.516 MMLV-II K348E 34.533 0.432 MMLV-II K348R 29.703 0.225 MMLV-II L198A 31.900 0.054 MMLV-II L198E 34.193 0.167 MMLV-II L198R 30.819 0.077 MMLV-II L280A 35.724 0.175 MMLV-II L280E 40.000 0.000 MMLV-II L280R 40.000 0.000 MMLV-II L352A 28.936 0.043 MMLV-II L352E 30.177 0.059 MMLV-II L352R 29.371 0.063 MMLV-II L357A 38.802 1.694 MMLV-II L357E 40.000 0.000 MMLV-II L357R 40.000 0.000 MMLV-II L82A 31.245 0.035 MMLV-II L82E 31.384 0.122 MMLV-II L82R 29.682 0.116 MMLV-II N335A 29.668 0.086 MMLV-II N335E 29.113 0.058 MMLV-II N335R 32.323 5.429 MMLV-II P76A 29.463 0.123 MMLV-II P76E 30.030 0.163 MMLV-II P76R 29.443 0.028 MMLV-II Q213A 29.833 0.223 MMLV-II Q213E 29.677 0.196 MMLV-II Q213R 29.704 0.053 MMLV-II Q299A 31.314 0.200 MMLV-II Q299E 28.652 0.149 MMLV-II Q299R 31.711 0.062 MMLV-II Q654A 29.415 0.117 MMLV-II Q654E 30.523 0.057 MMLV-II Q654R 29.523 0.052 MMLV-II R205A 29.140 0.138 MMLV-II R205E 29.356 0.179 MMLV-II R205K 29.162 0.206 MMLV-II R211A 29.491 0.025 MMLV-II R211E 30.049 0.205 MMLV-II R211K 30.196 0.147 MMLV-II R311A 31.237 0.425 MMLV-II R311E 40.000 0.000 MMLV-II R311K 29.857 0.091 MMLV-II R389A 32.173 0.151 MMLV-II R389E 32.717 0.105 MMLV-II R389K 31.944 0.166 MMLV-II R650A 29.734 0.060 MMLV-II R650E 31.012 0.074 MMLV-II R650K 29.404 0.094 MMLV-II R657A 31.470 0.133 MMLV-II R657E 32.785 0.145 MMLV-II R657K 29.468 0.274 MMLV-II S67A 29.268 0.090 MMLV-II S67E 30.157 0.254 MMLV-II S67R 27.274 0.054 MMLV-II T328A 40.000 0.000 MMLV-II T328E 37.699 1.627 MMLV-II T328R 37.169 0.848 MMLV-II T332A 29.219 0.075 MMLV-II T332E 29.714 0.057 MMLV-II T332R 30.462 0.130 MMLV-II V129A 29.305 0.077 MMLV-II V129E 31.188 0.181 MMLV-II V129R 30.383 0.081 MMLV-II V433A 30.483 0.059 MMLV-II V433E 30.106 0.144 MMLV-II V433R 29.297 0.457 MMLV-II V476A 31.295 0.244 MMLV-II V476E 34.664 0.364 MMLV-II V476R 31.223 0.166 MMLV-II Y271A 30.854 0.086 MMLV-II Y271E 28.620 0.068 MMLV-II Y271R 33.280 0.258 MMLV-IV 26.368 0.057

Example 8. Further Stacking of Reverse Transcriptase Mutants with Enhanced Activity

This example demonstrates the procedure used to stack the enhanced mutants found in Examples 6 and 7 to further improve the MMLV RTase's ability to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) compared to the base construct and previously found mutant MMLV RTase containing the following mutations: Q68R/Q79R/L99R/E282D. The stacked mutant MMLV RTases were cloned, overexpressed and purified as described in Examples 1 and 2 and tested as described in Examples 6 and 7. Both the two- and one-step reactions were analyzed and reported by Ct value (Tables 27-29). Six of the eight stacked mutant variants of MMLV RTase increased the overall activity and thermostability compared to the base construct, Q68R/Q79R/L99R/E282D/V433R, Q68R/Q79R/L99R/E282D/1593E, Q68R/Q79R/L99R/E282D/Q299E, Q68R/Q79R/L99R/E282D/T332E, Q68R/L82R/L99R/E282D and Q68R/Q79R/L82R/L99R/E282D. Sub sequentially, four of those six stacked mutant variants of MMLV RTase increased the overall activity and thermostability compared to the previously identified mutant RTase (Q68R/Q79R/L99R/E282D), Q68R/Q79R/L99R/E282D/1593E, Q68R/Q79R/L99R/E282D/Q299E, Q68R/L82R/L99R/E282D and Q68R/Q79R/L82R/L99R/E282D.

Following these stacked mutant variants, MMLV RTase mutations were stacked further to improve the ability of MMLV RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) as compared to the MMLV RTase base construct (RNase H minus construct). Eight MMLV RTase sextuple or more mutant variants were cloned as described in Example 1 and overexpressed and purified as in Example 5.

MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Temperatures were adjusted for both two-step and one-step reactions to 42/55 and 50/60° C., respectively. The two-step first strand synthesis buffer was modified from 50 mM Tris-hydrochloride, pH 8.3, 75 mM potassium chloride, 3 mM magnesium chloride and 10 mM DTT to 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.0, 10 mM magnesium acetate, 100 pg/ml bovine serum albumin and 10 mM DTT. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (Tables 27-29).

Four of the eleven MMLV RTase sextuple or more mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase stacked mutant variants, and almost all of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The four MMLV RTase mutant variants that were found to exhibit the highest overall activity were Q68R/Q79R/L99R/E282D/Q299E/V433R/V593E, Q68R2Q79R/L82R/L99R/E282D/Q299E/V433R/M593E, Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/593E, and Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E.

TABLE 27 Two-Step cDNA Synthesis by MMLV-RT stacked mutants using oligo dT priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 37.388 0.396 MMLV-II Q68R/Q79R/L99R/E282D/V433R 29.215 0.113 MMLV-II Q68R/Q79R/L99R/E282D/I593E 33.563 0.118 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 31.902 0.169 MMLV-II Q68R/Q79R/L99R/E282D/T332E 33.988 0.108 MMLV-II Q68R/Q79R/L99R/L280R 40.000 0.000 MMLV-II Q68R/Q79R/L99R/L280R/E282D 40.000 0.000 MMLV-II Q68R/L82R/L99R/E282D 39.259 1.047 MMLV-II Q68R/Q79R/L82R/L99R/E282D 30.623 0.076 MMLV-IV 25.880 0.023

TABLE 28 Two-Step cDNA Synthesis by MMLV-RT stacked mutants using random priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 36.638 1.014 MMLV-II Q68R/Q79R/L99R/E282D/V433R 40.000 0.000 MMLV-II Q68R/Q79R/L99R/E282D/I593E 32.331 0.111 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 30.430 0.154 MMLV-II Q68R/Q79R/L99R/E282D/T332E 33.720 0.266 MMLV-II Q68R/Q79R/L99R/L280R 40.000 0.000 MMLV-II Q68R/Q79R/L99R/L280R/E282D 40.000 0.000 MMLV-II Q68R/L82R/L99R/E282D 35.325 0.422 MMLV-II Q68R/Q79R/L82R/L99R/E282D 31.928 0.177 MMLV-IV 25.840 0.049

TABLE 29 One-Step cDNA Synthesis by MMLV-RT stacked mutants by gene specific priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 33.027 0.048 MMLV-II Q68R/Q79R/L99R/E282D/V433R 29.937 0.040 MMLV-II Q68R/Q79R/L99R/E282D/I593E 28.724 0.081 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 29.341 0.022 MMLV-II Q68R/Q79R/L99R/E282D/T332E 30.330 0.036 MMLV-II Q68R/Q79R/L99R/L280R 40.000 0.000 MMLV-II Q68R/Q79R/L99R/L280R/E282D 40.000 0.000 MMLV-II Q68R/L82R/L99R/E282D 30.559 0.045 MMLV-II Q68R/Q79R/L82R/L99R/E282D 30.097 0.033 MMLV-IV 28.975 0.012 a. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA Over a Wide Range of Temperatures

MMLV RTase base construct MMLV RTase mutant variants evaluated as described in Example 5. Oligo-dT or random hexamer priming conditions and reaction temperatures were adjusted for the two-step reactions and RTase concentration was normalized to 31 nM. The two-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see tables 25 and 26)

Five MMLV RTase mutants were found to exhibit high overall activity as compared to the MMLV RTase base construct over a wide range of temperatures, spanning from 37.0 to 51° C., regardless of which priming method used. All of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The five MMLV RTase mutant variants that were found to exhibit the highest overall activity at a wide range of temperatures were Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E, Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E, Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E and Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E

TABLE 30 Two-Step cDNA synthesis by MMLV RT quadruple and more mutants by Oligo-dT priming. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature of Ct Ct MMLV RT Mutant Reaction (° C.) Mean SD MMLV-II 37.0 26.340 0.033 MMLV-II 37.8 26.130 0.061 MMLV-II 39.5 25.830 0.014 MMLV-II 42.0 25.753 0.041 MMLV-II 45.2 25.632 0.077 MMLV-II 47.8 25.935 0.026 MMLV-II 49.2 26.478 0.042 MMLV-II 50.0 29.461 0.120 MMLV-II 51.0 29.430 0.098 MMLV-II 51.9 31.123 0.066 MMLV-II 53.8 33.632 0.073 MMLV-II 56.5 36.499 0.385 MMLV-II 59.9 37.158 0.427 MMLV-II 62.6 37.464 0.440 MMLV-II 64.2 37.082 0.022 MMLV-II 65.0 37.518 0.370 MMLV-II Q68R/Q79R/L99R/E282D 37.0 25.688 0.031 MMLV-II Q68R/Q79R/L99R/E282D 37.8 25.734 0.032 MMLV-II Q68R/Q79R/L99R/E282D 39.5 25.613 0.040 MMLV-II Q68R/Q79R/L99R/E282D 42.0 25.528 0.032 MMLV-II Q68R/Q79R/L99R/E282D 45.2 25.525 0.029 MMLV-II Q68R/Q79R/L99R/E282D 47.8 25.471 0.105 MMLV-II Q68R/Q79R/L99R/E282D 49.2 25.491 0.047 MMLV-II Q68R/Q79R/L99R/E282D 50.0 25.608 0.061 MMLV-II Q68R/Q79R/L99R/E282D 51.0 25.679 0.006 MMLV-II Q68R/Q79R/L99R/E282D 51.9 25.969 0.032 MMLV-II Q68R/Q79R/L99R/E282D 53.8 27.251 0.053 MMLV-II Q68R/Q79R/L99R/E282D 56.5 33.619 0.195 MMLV-II Q68R/Q79R/L99R/E282D 59.9 36.635 0.059 MMLV-II Q68R/Q79R/L99R/E282D 62.6 36.929 0.500 MMLV-II Q68R/Q79R/L99R/E282D 64.2 37.515 0.478 MMLV-II Q68R/Q79R/L99R/E282D 65.0 37.107 0.285 MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.0 26.133 0.054 MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.8 26.029 0.012 MMLV-II Q68R/Q79R/L99R/E282D/I593E 39.5 25.850 0.047 MMLV-II Q68R/Q79R/L99R/E282D/I593E 42.0 25.793 0.012 MMLV-II Q68R/Q79R/L99R/E282D/I593E 45.2 25.614 0.018 MMLV-II Q68R/Q79R/L99R/E282D/I593E 47.8 25.658 0.005 MMLV-II Q68R/Q79R/L99R/E282D/I593E 49.2 25.663 0.024 MMLV-II Q68R/Q79R/L99R/E282D/I593E 50.0 25.791 0.041 MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.0 25.877 0.067 MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.9 26.602 0.038 MMLV-II Q68R/Q79R/L99R/E282D/I593E 53.8 29.535 0.086 MMLV-II Q68R/Q79R/L99R/E282D/I593E 56.5 35.912 0.439 MMLV-II Q68R/Q79R/L99R/E282D/I593E 59.9 37.158 0.566 MMLV-II Q68R/Q79R/L99R/E282D/I593E 62.6 37.187 0.158 MMLV-II Q68R/Q79R/L99R/E282D/I593E 64.2 37.958 0.236 MMLV-II Q68R/Q79R/L99R/E282D/I593E 65.0 36.861 0.416 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.0 26.106 0.070 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.8 26.024 0.092 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 39.5 25.830 0.122 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 42.0 25.788 0.025 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 45.2 25.634 0.022 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 47.8 25.681 0.016 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 49.2 25.684 0.029 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 50.0 25.743 0.096 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.0 25.870 0.003 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.9 26.301 0.033 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 53.8 28.283 0.036 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 56.5 34.732 0.445 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 59.9 36.947 0.407 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 62.6 37.140 0.280 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 64.2 37.403 0.205 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 65.0 37.347 0.438 MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.0 25.961 0.170 MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.8 26.065 0.085 MMLV-II Q68R/Q79R/L82R/L99R/E282D 39.5 25.909 0.028 MMLV-II Q68R/Q79R/L82R/L99R/E282D 42.0 25.802 0.055 MMLV-II Q68R/Q79R/L82R/L99R/E282D 45.2 25.632 0.087 MMLV-II Q68R/Q79R/L82R/L99R/E282D 47.8 25.728 0.065 MMLV-II Q68R/Q79R/L82R/L99R/E282D 49.2 25.612 0.165 MMLV-II Q68R/Q79R/L82R/L99R/E282D 50.0 25.795 0.038 MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.0 25.830 0.009 MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.9 26.477 0.037 MMLV-II Q68R/Q79R/L82R/L99R/E282D 53.8 28.496 0.040 MMLV-II Q68R/Q79R/L82R/L99R/E282D 56.5 34.329 0.177 MMLV-II Q68R/Q79R/L82R/L99R/E282D 59.9 36.564 0.315 MMLV-II Q68R/Q79R/L82R/L99R/E282D 62.6 37.152 0.322 MMLV-II Q68R/Q79R/L82R/L99R/E282D 64.2 37.340 0.585 MMLV-II Q68R/Q79R/L82R/L99R/E282D 65.0 38.351 1.016 MMLV-II 37.0 25.853 0.057 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.8 25.898 0.016 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 39.5 25.716 0.093 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 42.0 25.669 0.064 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 45.2 25.643 0.056 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 47.8 25.680 0.016 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 49.2 25.663 0.057 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 50.0 25.708 0.045 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.0 25.557 0.025 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.9 26.015 0.125 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 53.8 27.812 0.048 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 56.5 34.073 0.217 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 59.9 36.512 0.168 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 62.6 37.182 0.167 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 64.2 37.239 0.291 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 65.0 36.573 0.232 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.0 25.789 0.075 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.8 25.784 0.103 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 39.5 25.714 0.025 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 42.0 25.713 0.027 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 45.2 25.690 0.030 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 47.8 25.662 0.026 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 49.2 25.713 0.021 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 50.0 25.551 0.092 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.0 25.561 0.107 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.9 25.975 0.125 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 53.8 27.556 0.023 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 56.5 33.934 0.249 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 59.9 36.473 0.285 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 62.6 37.411 0.377 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 64.2 37.656 0.478 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 65.0 37.950 1.451 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.0 25.788 0.028 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 37.8 25.680 0.229 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 39.5 25.794 0.051 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 42.0 25.415 0.270 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 45.2 25.631 0.047 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 47.8 25.672 0.027 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 49.2 25.792 0.045 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 50.0 25.759 0.022 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 51.0 25.852 0.015 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 51.9 26.425 0.033 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 53.8 29.964 0.023 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 56.5 36.532 0.113 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 59.9 38.246 0.608 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 62.6 37.333 0.446 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 64.2 37.223 0.212 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 65.0 36.930 0.527 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 37.0 25.863 0.014 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 37.8 25.649 0.036 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 39.5 25.573 0.057 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 42.0 25.453 0.023 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 45.2 25.447 0.083 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 47.8 25.413 0.061 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 49.2 25.542 0.035 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 50.0 25.567 0.060 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 51.0 25.741 0.093 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 51.9 26.231 0.225 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 53.8 28.556 0.142 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 56.5 35.202 0.208 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 59.9 36.991 0.419 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 62.6 37.168 0.463 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 64.2 37.670 0.410 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 65.0 37.680 0.273 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E

TABLE 31 Two-Step cDNA synthesis by MMLV RT quadruple and more mutants by Random priming. Data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature of Reaction Ct Ct MMLV RT Mutant (° C.) Mean SD MMLV-II 37.0 26.365 0.066 MMLV-II 37.8 26.390 0.006 MMLV-II 39.5 25.939 0.016 MMLV-II 42.0 25.798 0.029 MMLV-II 45.2 25.849 0.064 MMLV-II 47.8 26.647 0.050 MMLV-II 49.2 28.326 0.028 MMLV-II 50.0 29.340 0.010 MMLV-II 51.0 30.684 0.099 MMLV-II 51.9 32.462 0.163 MMLV-II 53.8 33.855 0.307 MMLV-II 56.5 35.376 0.461 MMLV-II 59.9 36.098 0.481 MMLV-II 62.6 36.391 0.367 MMLV-II 64.2 36.442 0.547 MMLV-II 65.0 35.871 0.301 MMLV-II Q68R/Q79R/L99R/E282D 37.0 25.699 0.009 MMLV-II Q68R/Q79R/L99R/E282D 37.8 25.674 0.038 MMLV-II Q68R/Q79R/L99R/E282D 39.5 25.594 0.029 MMLV-II Q68R/Q79R/L99R/E282D 42.0 25.496 0.016 MMLV-II Q68R/Q79R/L99R/E282D 45.2 25.431 0.011 MMLV-II Q68R/Q79R/L99R/E282D 47.8 25.420 0.036 MMLV-II Q68R/Q79R/L99R/E282D 49.2 25.481 0.023 MMLV-II Q68R/Q79R/L99R/E282D 50.0 25.646 0.035 MMLV-II Q68R/Q79R/L99R/E282D 51.0 25.979 0.012 MMLV-II Q68R/Q79R/L99R/E282D 51.9 26.591 0.053 MMLV-II Q68R/Q79R/L99R/E282D 53.8 28.345 0.091 MMLV-II Q68R/Q79R/L99R/E282D 56.5 32.976 0.109 MMLV-II Q68R/Q79R/L99R/E282D 59.9 34.407 0.158 MMLV-II Q68R/Q79R/L99R/E282D 62.6 35.130 0.014 MMLV-II Q68R/Q79R/L99R/E282D 64.2 34.866 0.258 MMLV-II Q68R/Q79R/L99R/E282D 65.0 35.317 0.299 MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.0 26.079 0.036 MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.8 25.951 0.015 MMLV-II Q68R/Q79R/L99R/E282D/I593E 39.5 25.801 0.055 MMLV-II Q68R/Q79R/L99R/E282D/I593E 42.0 25.602 0.087 MMLV-II Q68R/Q79R/L99R/E282D/I593E 45.2 25.424 0.038 MMLV-II Q68R/Q79R/L99R/E282D/I593E 47.8 25.520 0.011 MMLV-II Q68R/Q79R/L99R/E282D/I593E 49.2 25.674 0.046 MMLV-II Q68R/Q79R/L99R/E282D/I593E 50.0 25.922 0.015 MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.0 26.351 0.014 MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.9 27.411 0.092 MMLV-II Q68R/Q79R/L99R/E282D/I593E 53.8 30.482 0.048 MMLV-II Q68R/Q79R/L99R/E282D/I593E 56.5 33.914 0.075 MMLV-II Q68R/Q79R/L99R/E282D/I593E 59.9 35.443 0.191 MMLV-II Q68R/Q79R/L99R/E282D/I593E 62.6 35.872 0.445 MMLV-II Q68R/Q79R/L99R/E282D/I593E 64.2 36.107 0.011 MMLV-II Q68R/Q79R/L99R/E282D/I593E 65.0 35.715 0.299 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.0 25.955 0.040 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.8 25.934 0.023 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 39.5 25.669 0.035 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 42.0 25.523 0.016 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 45.2 25.532 0.054 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 47.8 25.550 0.021 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 49.2 25.620 0.030 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 50.0 25.711 0.035 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.0 26.215 0.056 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.9 26.969 0.013 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 53.8 29.622 0.060 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 56.5 33.679 0.234 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 59.9 35.253 0.144 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 62.6 35.408 0.441 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 64.2 35.586 0.139 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 65.0 36.076 0.700 MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.0 25.884 0.012 MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.8 25.833 0.009 MMLV-II Q68R/Q79R/L82R/L99R/E282D 39.5 25.684 0.077 MMLV-II Q68R/Q79R/L82R/L99R/E282D 42.0 25.553 0.026 MMLV-II Q68R/Q79R/L82R/L99R/E282D 45.2 25.471 0.043 MMLV-II Q68R/Q79R/L82R/L99R/E282D 47.8 25.491 0.085 MMLV-II Q68R/Q79R/L82R/L99R/E282D 49.2 25.646 0.014 MMLV-II Q68R/Q79R/L82R/L99R/E282D 50.0 25.765 0.039 MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.0 26.365 0.044 MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.9 27.170 0.071 MMLV-II Q68R/Q79R/L82R/L99R/E282D 53.8 29.662 0.048 MMLV-II Q68R/Q79R/L82R/L99R/E282D 56.5 33.853 0.162 MMLV-II Q68R/Q79R/L82R/L99R/E282D 59.9 34.899 0.325 MMLV-II Q68R/Q79R/L82R/L99R/E282D 62.6 35.557 0.145 MMLV-II Q68R/Q79R/L82R/L99R/E282D 64.2 35.360 0.222 MMLV-II Q68R/Q79R/L82R/L99R/E282D 65.0 35.614 0.403 MMLV-II 37.0 25.706 0.031 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.8 25.757 0.101 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 39.5 25.435 0.036 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 42.0 25.417 0.025 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 45.2 25.425 0.023 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 47.8 25.401 0.049 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 49.2 25.467 0.009 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 50.0 25.516 0.056 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.0 25.880 0.039 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.9 26.348 0.064 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 53.8 28.506 0.018 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 56.5 32.812 0.242 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 59.9 34.123 0.163 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 62.6 35.108 0.027 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 64.2 34.796 0.171 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 65.0 34.999 0.064 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.0 25.711 0.080 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.8 25.916 0.224 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 39.5 25.665 0.052 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 42.0 25.527 0.016 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 45.2 25.504 0.065 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 47.8 25.437 0.070 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 49.2 25.555 0.065 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 50.0 25.571 0.028 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.0 25.854 0.029 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 51.9 26.259 0.057 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 53.8 28.329 0.053 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 56.5 32.962 0.212 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 59.9 34.072 0.446 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 62.6 34.931 0.205 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 64.2 34.626 0.169 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 65.0 35.085 0.230 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E MMLV-II 37.0 25.940 0.130 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 37.8 25.793 0.129 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 39.5 25.599 0.015 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 42.0 25.504 0.016 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 45.2 25.602 0.041 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 47.8 25.604 0.058 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 49.2 25.665 0.007 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 50.0 25.821 0.068 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 51.0 26.315 0.047 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 51.9 27.036 0.059 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 53.8 31.004 0.089 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 56.5 33.765 0.274 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 59.9 34.656 0.209 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 62.6 35.561 0.468 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 64.2 35.877 0.154 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 65.0 35.659 0.477 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E MMLV-II 37.0 25.780 0.046 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 37.8 25.652 0.026 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 39.5 25.641 0.037 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 42.0 25.507 0.005 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 45.2 25.484 0.067 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 47.8 25.438 0.027 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 49.2 25.534 0.022 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 50.0 25.755 0.085 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 51.0 25.981 0.027 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 51.9 26.242 0.052 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 53.8 29.146 0.069 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 56.5 33.138 0.159 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 59.9 34.551 0.152 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 62.6 35.186 0.322 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R /I593E MMLV-II 64.2 35.550 0.368 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E MMLV-II 65.0 35.459 0.295 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ I593E

Example 9: Extension of Reverse Transcriptase Single Mutants

The amino acid positions that enclosed the MMLV RTase single mutants identified in Examples 6 and 7 were further evaluated to include all possible amino acid substitutions at that position. The single mutants were cloned, overexpressed, and purified as described in Examples 1 and 2, and evaluated as described in Examples 6 and 7. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase double mutant variants were analyzed and reported by Ct output from the qPCR (Tables 32-34). Numerous single mutant MMLV RTase variants were found to exhibit an increase in the overall activity and thermostability as compared to the MMLV RTase base construct. The most prevalent among these were: L82F, L82K, L82T, L82Y, L280I, T332V, V433K, V433N, and I593W.

TABLE 32 Two-Step cDNA Synthesis by MMLV-RT single mutants using Oligo-dT priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 40.000 0.000 MMLV-II I593A 40.000 0.000 MMLV-II I593C 37.874 0.991 MMLV-II I593D 40.000 0.000 MMLV-II I593E 40.000 0.000 MMLV-II I593F 40.000 0.000 MMLV-II I593G 39.748 0.356 MMLV-II I593H 39.502 0.704 MMLV-II I593K 40.000 0.000 MMLV-II I593L 38.994 1.423 MMLV-II I593M 39.383 0.873 MMLV-II I593N 40.000 0.000 MMLV-II I593P 40.000 0.000 MMLV-II I593Q 40.000 0.000 MMLV-II I593R 40.000 0.000 MMLV-II I593S 39.614 0.545 MMLV-II I593T 37.709 0.520 MMLV-II I593V 40.000 0.000 MMLV-II I593W 30.504 0.073 MMLV-II I593Y 40.000 0.000 MMLV-II L280A 40.000 0.000 MMLV-II L280C 40.000 0.000 MMLV-II L280D 40.000 0.000 MMLV-II L280E 40.000 0.000 MMLV-II L280F 40.000 0.000 MMLV-II L280G 40.000 0.000 MMLV-II L280H 40.000 0.000 MMLV-II L280I 30.951 0.076 MMLV-II L280K 40.000 0.000 MMLV-II L280M 40.000 0.000 MMLV-II L280N 39.727 0.386 MMLV-II L280P 40.000 0.000 MMLV-II L280Q 40.000 0.000 MMLV-II L280R 39.994 0.009 MMLV-II L280S 40.000 0.000 MMLV-II L280T 40.000 0.000 MMLV-II L280V 37.749 0.142 MMLV-II L280W 40.000 0.000 MMLV-II L280Y 40.000 0.000 MMLV-II L82A 40.000 0.000 MMLV-II L82C 39.565 0.615 MMLV-II L82D 40.000 0.000 MMLV-II L82E 40.000 0.000 MMLV-II L82F 39.347 0.924 MMLV-II L82G 40.000 0.000 MMLV-II L82H 40.000 0.000 MMLV-II L82I 40.000 0.000 MMLV-II L82K 37.136 0.593 MMLV-II L82M 38.649 1.260 MMLV-II L82N 40.000 0.000 MMLV-II L82P 40.000 0.000 MMLV-II L82Q 39.098 1.275 MMLV-II L82R 40.000 0.000 MMLV-II L82S 39.346 0.925 MMLV-II L82T 38.695 1.845 MMLV-II L82V 38.047 1.381 MMLV-II L82W 37.151 0.308 MMLV-II L82Y 35.014 0.421 MMLV-II Q299A 40.000 0.000 MMLV-II Q299C 40.000 0.000 MMLV-II Q299D 40.000 0.000 MMLV-II Q299E 39.061 1.328 MMLV-II Q299F 40.000 0.000 MMLV-II Q299G 40.000 0.000 MMLV-II Q299H 39.398 0.852 MMLV-II Q299I 39.183 1.155 MMLV-II Q299K 40.000 0.000 MMLV-II Q299L 39.474 0.743 MMLV-II Q299M 40.000 0.000 MMLV-II Q299N 40.000 0.000 MMLV-II Q299P 40.000 0.000 MMLV-II Q299R 40.000 0.000 MMLV-II Q299S 40.000 0.000 MMLV-II Q299T 40.000 0.000 MMLV-II Q299V 40.000 0.000 MMLV-II Q299W 40.000 0.000 MMLV-II Q299Y 40.000 0.000 MMLV-II T332A 39.087 1.291 MMLV-II T332C 38.956 1.476 MMLV-II T332D 40.000 0.000 MMLV-II T332E 39.554 0.631 MMLV-II T332F 40.000 0.000 MMLV-II T332G 37.321 2.009 MMLV-II T332H 39.215 1.110 MMLV-II T332I 39.344 0.927 MMLV-II T332K 40.000 0.000 MMLV-II T332L 40.000 0.000 MMLV-II T332M 37.775 1.632 MMLV-II T332N 37.326 0.834 MMLV-II T332P 40.000 0.000 MMLV-II T332Q 39.509 0.694 MMLV-II T332R 39.588 0.582 MMLV-II T332S 39.765 0.332 MMLV-II T332V 36.977 0.384 MMLV-II T332W 40.000 0.000 MMLV-II T332Y 40.000 0.000 MMLV-II V433A 40.000 0.000 MMLV-II V433C 37.504 0.682 MMLV-II V433D 40.000 0.000 MMLV-II V433E 35.189 0.336 MMLV-II V433F 39.379 0.878 MMLV-II V433G 39.482 0.732 MMLV-II V433H 40.000 0.000 MMLV-II V433I 39.781 0.310 MMLV-II V433K 35.770 0.623 MMLV-II V433L 39.015 0.744 MMLV-II V433M 39.119 1.247 MMLV-II V433N 33.981 0.185 MMLV-II V433P 40.000 0.000 MMLV-II V433Q 40.000 0.000 MMLV-II V433R 37.230 1.247 MMLV-II V433S 37.850 0.846 MMLV-II V433T 37.564 1.895 MMLV-II V433W 37.770 1.622 MMLV-II V433Y 40.000 0.000 MMLV-IV 26.102 0.033

TABLE 33 Two-Step cDNA Synthesis by MMLV-RT single mutants using random priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Standard MMLV-RT Variant Ct Mean Deviation MMLV-II 40.000 0.000 MMLV-II I593A 40.000 0.000 MMLV-II I593C 40.000 0.000 MMLV-II I593D 39.992 0.012 MMLV-II I593E 40.000 0.000 MMLV-II I593F 39.189 1.147 MMLV-II I593G 40.000 0.000 MMLV-II I593H 40.000 0.000 MMLV-II I593K 40.000 0.000 MMLV-II I593L 40.000 0.000 MMLV-II I593M 40.000 0.000 MMLV-II I593N 40.000 0.000 MMLV-II I593P 40.000 0.000 MMLV-II I593Q 39.201 0.853 MMLV-II I593R 38.928 1.516 MMLV-II I593S 39.025 1.379 MMLV-II I593T 38.385 1.227 MMLV-II I593V 39.574 0.603 MMLV-II I593W 32.572 0.054 MMLV-II I593Y 40.000 0.000 MMLV-II L280A 40.000 0.000 MMLV-II L280C 40.000 0.000 MMLV-II L280D 40.000 0.000 MMLV-II L280E 40.000 0.000 MMLV-II L280F 40.000 0.000 MMLV-II L280G 40.000 0.000 MMLV-II L280H 40.000 0.000 MMLV-II L280I 34.152 0.276 MMLV-II L280K 40.000 0.000 MMLV-II L280M 39.973 0.038 MMLV-II L280N 40.000 0.000 MMLV-II L280P 40.000 0.000 MMLV-II L280Q 40.000 0.000 MMLV-II L280R 40.000 0.000 MMLV-II L280S 40.000 0.000 MMLV-II L280T 40.000 0.000 MMLV-II L280V 39.260 1.046 MMLV-II L280W 40.000 0.000 MMLV-II L280Y 40.000 0.000 MMLV-II L82A 40.000 0.000 MMLV-II L82C 40.000 0.000 MMLV-II L82D 40.000 0.000 MMLV-II L82E 39.672 0.463 MMLV-II L82F 36.854 0.708 MMLV-II L82G 40.000 0.000 MMLV-II L82H 37.705 0.557 MMLV-II L82I 39.231 1.087 MMLV-II L82K 39.437 0.443 MMLV-II L82M 40.000 0.000 MMLV-II L82N 40.000 0.000 MMLV-II L82P 40.000 0.000 MMLV-II L82Q 40.000 0.000 MMLV-II L82R 38.595 1.191 MMLV-II L82S 40.000 0.000 MMLV-II L82T 38.449 1.192 MMLV-II L82V 39.438 0.795 MMLV-II L82W 39.178 1.163 MMLV-II L82Y 36.758 0.962 MMLV-II Q299A 40.000 0.000 MMLV-II Q299C 40.000 0.000 MMLV-II Q299D 38.003 1.414 MMLV-II Q299E 39.338 0.936 MMLV-II Q299F 40.000 0.000 MMLV-II Q299G 40.000 0.000 MMLV-II Q299H 40.000 0.000 MMLV-II Q299I 39.850 0.212 MMLV-II Q299K 40.000 0.000 MMLV-II Q299L 40.000 0.000 MMLV-II Q299M 40.000 0.000 MMLV-II Q299N 40.000 0.000 MMLV-II Q299P 40.000 0.000 MMLV-II Q299R 40.000 0.000 MMLV-II Q299S 40.000 0.000 MMLV-II Q299T 40.000 0.000 MMLV-II Q299V 40.000 0.000 MMLV-II Q299W 40.000 0.000 MMLV-II Q299Y 40.000 0.000 MMLV-II T332A 39.814 0.264 MMLV-II T332C 40.000 0.000 MMLV-II T332D 40.000 0.000 MMLV-II T332E 40.000 0.000 MMLV-II T332F 40.000 0.000 MMLV-II T332G 38.897 1.560 MMLV-II T332H 40.000 0.000 MMLV-II T332I 40.000 0.000 MMLV-II T332K 40.000 0.000 MMLV-II T332L 38.169 2.589 MMLV-II T332M 37.410 1.906 MMLV-II T332N 38.983 1.362 MMLV-II T332P 39.046 1.350 MMLV-II T332Q 40.000 0.000 MMLV-II T332R 40.000 0.000 MMLV-II T332S 40.000 0.000 MMLV-II T332V 38.650 1.326 MMLV-II T332W 40.000 0.000 MMLV-II T332Y 40.000 0.000 MMLV-II V433A 40.000 0.000 MMLV-II V433C 37.605 0.184 MMLV-II V433D 40.000 0.000 MMLV-II V433E 34.693 0.193 MMLV-II V433F 40.000 0.000 MMLV-II V433G 40.000 0.000 MMLV-II V433H 40.000 0.000 MMLV-II V433I 39.792 0.294 MMLV-II V433K 35.725 0.464 MMLV-II V433L 40.000 0.000 MMLV-II V433M 40.000 0.000 MMLV-II V433N 34.604 0.554 MMLV-II V433P 40.000 0.000 MMLV-II V433Q 38.844 1.001 MMLV-II V433R 38.817 0.839 MMLV-II V433S 38.202 1.372 MMLV-II V433T 37.573 0.623 MMLV-II V433W 37.611 1.690 MMLV-II V433Y 40.000 0.000 MMLV-IV 26.053 0.098

TABLE 34 One-Step cDNA Synthesis by MMLV-RT single mutants by gene specific priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Ct Ct Standard MMLV-RT Variant Mean Deviation MMLV-II 32.775 0.189 MMLV-II I593A 32.438 0.209 MMLV-II I593C 32.680 0.053 MMLV-II I593D 31.775 0.237 MMLV-II I593E 30.635 0.048 MMLV-II I593F 30.411 0.008 MMLV-II I593G 30.904 0.098 MMLV-II I593H 29.686 0.131 MMLV-II I593K 31.832 0.259 MMLV-II I593L 32.289 0.273 MMLV-II I593M 32.162 0.078 MMLV-II I593N 31.410 0.251 MMLV-II I593P 34.728 0.201 MMLV-II I593Q 31.609 0.032 MMLV-II I593R 31.144 0.133 MMLV-II I593S 30.548 0.247 MMLV-II I593T 29.572 0.236 MMLV-II I593V 30.673 0.142 MMLV-II I593W 28.179 0.092 MMLV-II I593Y 30.858 0.067 MMLV-II L280A 36.160 0.729 MMLV-II L280C 32.097 0.261 MMLV-II L280D 40.000 0.000 MMLV-II L280E 39.115 1.251 MMLV-II L280F 34.573 0.371 MMLV-II L280G 40.000 0.000 MMLV-II L280H 37.255 0.322 MMLV-II L280I 29.267 1.032 MMLV-II L280K 34.274 0.095 MMLV-II L280M 32.746 0.223 MMLV-II L280N 39.677 0.457 MMLV-II L280P 33.045 0.095 MMLV-II L280Q 39.190 1.145 MMLV-II L280R 40.000 0.000 MMLV-II L280S 40.000 0.000 MMLV-II L280T 37.074 0.325 MMLV-II L280V 30.461 0.052 MMLV-II L280W 40.000 0.000 MMLV-II L280Y 40.000 0.000 MMLV-II L82A 31.729 0.308 MMLV-II L82C 31.131 0.192 MMLV-II L82D 34.280 0.227 MMLV-II L82E 32.973 0.430 MMLV-II L82F 29.760 0.030 MMLV-II L82G 33.066 0.217 MMLV-II L82H 30.098 0.078 MMLV-II L82I 31.605 0.083 MMLV-II L82K 29.258 0.015 MMLV-II L82M 30.280 0.027 MMLV-II L82N 33.074 0.323 MMLV-II L82P 38.754 1.762 MMLV-II L82Q 32.001 0.164 MMLV-II L82R 30.208 0.128 MMLV-II L82S 31.841 0.231 MMLV-II L82T 28.908 0.044 MMLV-II L82V 29.533 0.057 MMLV-II L82W 29.580 0.056 MMLV-II L82Y 28.934 0.073 MMLV-II Q299A 31.113 0.138 MMLV-II Q299C 35.953 0.542 MMLV-II Q299D 32.292 0.080 MMLV-II Q299E 31.663 0.027 MMLV-II Q299F 36.143 0.317 MMLV-II Q299G 31.929 0.131 MMLV-II Q299H 32.387 0.133 MMLV-II Q299I 37.763 1.582 MMLV-II Q299K 32.326 0.096 MMLV-II Q299L 34.807 0.180 MMLV-II Q299M 32.514 0.375 MMLV-II Q299N 34.040 0.186 MMLV-II Q299P 39.460 0.764 MMLV-II Q299R 33.044 0.354 MMLV-II Q299S 33.438 0.256 MMLV-II Q299T 35.093 0.926 MMLV-II Q299V 35.114 1.045 MMLV-II Q299W 38.998 1.417 MMLV-II Q299Y 39.055 1.336 MMLV-II T332A 30.528 0.084 MMLV-II T332C 30.785 0.135 MMLV-II T332D 33.310 0.348 MMLV-II T332E 32.711 0.106 MMLV-II T332F 33.201 0.179 MMLV-II T332G 30.424 0.054 MMLV-II T332H 31.913 0.306 MMLV-II T332I 32.072 0.115 MMLV-II T332K 31.591 0.082 MMLV-II T332L 34.011 0.133 MMLV-II T332M 29.039 0.164 MMLV-II T332N 29.500 0.135 MMLV-II T332P 33.976 0.272 MMLV-II T332Q 31.599 0.041 MMLV-II T332R 32.950 0.130 MMLV-II T332S 31.003 0.341 MMLV-II T332V 29.835 0.061 MMLV-II T332W 35.431 0.099 MMLV-II T332Y 33.384 0.164 MMLV-II V433A 30.757 0.105 MMLV-II V433C 29.901 0.305 MMLV-II V433D 34.152 0.170 MMLV-II V433E 28.868 0.011 MMLV-II V433F 31.529 0.009 MMLV-II V433G 33.663 0.412 MMLV-II V433H 31.811 0.069 MMLV-II V433I 30.460 0.071 MMLV-II V433K 30.040 0.109 MMLV-II V433L 31.758 0.063 MMLV-II V433M 30.791 0.095 MMLV-II V433N 28.566 0.074 MMLV-II V433P 37.436 1.824 MMLV-II V433Q 30.586 0.104 MMLV-II V433R 30.773 0.080 MMLV-II V433S 29.768 0.074 MMLV-II V433T 29.096 0.107 MMLV-II V433W 29.130 0.064 MMLV-II V433Y 32.676 0.279 MMLV-IV 25.979 0.043

TABLE 35 Two-Step cDNA Synthesis by MMLV-RT stacked mutants using oligo dT priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature Ct Ct Standard MMLV-RT Variant (° C.) Mean Deviation MMLV-II 42 25.207 0.025 MMLV-II 55 28.180 0.022 MMLV-II 42 25.287 0.068 Q68R/Q79R/L99R/E282D 55 26.442 0.044 MMLV-II 42 25.344 0.065 Q68R/Q79R/L99R/E282D/V433R 55 26.586 0.077 MMLV-II 42 25.266 0.112 Q68R/Q79R/L99R/E282D/I593E 55 27.389 0.069 MMLV-II 42 25.357 0.087 Q68R/Q79R/L99R/E282D/Q299E 55 26.953 0.034 MMLV-II 42 25.394 0.011 Q68R/Q79R/L82R/L99R/E282D 55 27.171 0.028 MMLV-II 42 25.371 0.061 Q68R/Q79R/L99R/E282D/Q299E/ 55 26.689 0.068 I593E MMLV-II 42 25.258 0.035 Q68R/Q79R/L82R/L99R/E282D/ 55 26.979 0.034 Q299E/I593E MMLV-II 42 25.171 0.006 Q68R/Q79R/L99R/E282D/Q299E/ 55 26.299 0.025 V433R/I593E MMLV-II 42 25.146 0.052 Q68R/Q79R/L82R/L99R/E282D/ 55 26.320 0.036 Q299E/V433R/I593E MMLV-II 42 25.176 0.044 Q68R/Q79R/L82R/L99R/E282D/ 55 26.750 0.040 Q299E/T332E/I593E MMLV-II 42 25.110 0.046 Q68R/Q79R/L82R/L99R/E282D/ 55 26.587 0.049 Q299E/T332E/V433R/I593E MMLV-IV 42 25.184 0.025 MMLV-IV 55 25.153 0.037 SuperScript-IV 42 25.082 0.073 SuperScript-IV 55 25.080 0.047

TABLE 36 Two-Step cDNA Synthesis by MMLV-RT stacked mutants using random priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature Ct Ct Standard MMLV-RT Variant (C) Mean Deviation MMLV-II 42 25.264 0.019 MMLV-II 55 28.443 0.014 MMLV-II Q68R/Q79R/L99R/E282D 42 25.399 0.040 55 26.484 0.072 MMLV-II Q68R/Q79R/L99R/E282D/V433R 42 25.324 0.063 55 26.794 0.065 MMLV-II Q68R/Q79R/L99R/E282D/I593E 42 25.278 0.025 55 27.616 0.058 MMLV-II Q68R/Q79R/L99R/E282D/Q299E 42 25.281 0.079 55 27.148 0.025 MMLV-II Q68R/Q79R/L82R/L99R/E282D 42 25.279 0.053 55 27.243 0.008 MMLV-II Q68R/Q79R/L99R/E282D/Q299E/I593E 42 25.409 0.065 55 26.704 0.066 MMLV-II 42 25.581 0.062 Q68R/Q79R/L82R/L99R/E282D/Q299E/I593E 55 26.605 0.028 MMLV-II 42 25.355 0.158 Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 55 26.305 0.066 MMLV-II 42 25.418 0.120 Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E 55 26.403 0.055 MMLV-II 42 25.374 0.115 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E 55 26.747 0.065 MMLV-II 42 25.426 0.082 Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ 55 26.481 0.017 I593E MMLV-IV 42 25.394 0.162 MMLV-IV 55 25.185 0.022 SuperScript-IV 42 25.299 0.132 SuperScript-IV 55 25.214 0.021

TABLE 37 One-Step cDNA Synthesis by MMLV-RT stacked mutants by gene specific priming. The data was generated via qPCR human normalizer assay and data is reported by Ct value. Temperature Concentration Ct Ct Standard MMLV-RT Variant (° C.) of RT (nM) Mean Deviation MMLV-II 50 0.28 26.401 0.022 1.4 24.701 0.061 7.0 24.664 0.007 60 0.28 31.134 0.205 1.4 28.109 0.042 7.0 27.644 0.061 MMLV-II 50 0.28 25.171 0.046 Q68R/Q79R/L99R/ 1.4 24.440 0.037 E282D 7.0 24.406 0.010 60 0.28 28.848 0.114 1.4 25.905 0.066 7.0 25.618 0.057 MMLV-II 50 0.28 24.967 0.068 Q68R/Q79R/L99R/ 1.4 24.386 0.015 E282D/V433R 7.0 24.433 0.079 60 0.28 28.516 0.051 1.4 25.803 0.063 7.0 25.620 0.035 MMLV-II 50 0.28 24.660 0.053 Q68R/Q79R/L99R/ 1.4 24.377 0.028 E282D/I593E 7.0 24.355 0.021 60 0.28 27.488 0.074 1.4 25.413 0.049 7.0 25.209 0.136 MMLV-II 50 0.28 25.044 0.094 Q68R/Q79R/L99R/ 1.4 24.422 0.023 E282D/Q299E 7.0 24.528 0.055 60 0.28 28.818 0.137 1.4 25.953 0.082 7.0 25.754 0.098 MMLV-II 50 0.28 25.014 0.152 Q68R/Q79R/L82R/ 1.4 24.467 0.020 L99R/E282D 7.0 24.507 0.046 60 0.28 28.743 0.076 1.4 26.662 0.012 7.0 25.883 0.022 MMLV-II 50 0.28 24.771 0.027 Q68R/Q79R/L99R/ 1.4 24.501 0.008 E282D/Q299E/I593E 7.0 24.485 0.087 60 0.28 27.721 0.057 1.4 25.836 0.030 7.0 25.199 0.016 MMLV-II 50 0.28 24.777 0.029 Q68R/Q79R/L82R/ 1.4 24.432 0.033 L99R/E282D/Q299E/ 7.0 24.435 0.024 I593E 60 0.28 27.854 0.035 1.4 25.613 0.028 7.0 25.072 0.030 MMLV-II 50 0.28 24.550 0.003 Q68R/Q79R/L99R/ 1.4 24.333 0.033 E282D/Q299E/V433R/ 7.0 24.345 0.030 I593E 60 0.28 26.399 0.051 1.4 25.236 0.040 7.0 25.105 0.050 MMLV-II 50 0.28 24.562 0.047 Q68R/Q79R/L82R/ 1.4 24.350 0.039 L99R/E282D/Q299E/ 7.0 24.302 0.015 V433R/I593E 60 0.28 26.459 0.022 1.4 25.247 0.069 7.0 25.001 0.050 MMLV-II 50 0.28 24.614 0.047 Q68R/Q79R/L82R/ 1.4 24.420 0.051 L99R/E282D/Q299E/ 7.0 24.361 0.021 T332E/I593E 60 0.28 26.769 0.089 1.4 25.609 0.041 7.0 25.348 0.043 MMLV-II 50 0.28 24.594 0.075 Q68R/Q79R/L82R/ 1.4 24.402 0.045 L99R/E282D/Q299E/ 7.0 24.291 0.057 T332E/V433R/I593E 60 0.28 26.591 0.018 1.4 25.517 0.048 7.0 25.193 0.027 MMLV-IV 50 0.28 24.397 0.091 1.4 24.303 0.062 7.0 24.189 0.039 60 0.28 25.807 0.045 1.4 25.180 0.037 7.0 24.625 0.011 SuperScript-IV 50 0.28 24.743 0.049 1.4 24.213 0.017 7.0 24.008 0.036 60 0.28 26.124 0.103 1.4 24.681 0.070 7.0 24.180 0.082

TABLE 38 Sequences of quadruple or more mutant MMLV RTase variants. SEQ ID NO: Construct Construct Sequence (AA) 686 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG V433R TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 687 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG I593E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 688 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG Q299E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYINSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 689 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG T332E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGELFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 690 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q681/Q791/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/L280R RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWR TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 691 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/L280R/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG E282D TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWR TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 692 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/L82R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D RLGIKPHIQRLRDQGILVPCQSPWNTPLRPVKKPG TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 693 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG E282D TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 694 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG Q299E/I593E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 695 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYTNSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 696 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG Q299E/V433R/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 697 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP V433R/I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 698 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP T332E/I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGELFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF 699 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP T332E/V433R/ PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP I593E EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP GFAEMAAPLYPLTKTGELFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP DTSTLLIENSSPYTSEHF

BIBLIOGRAPHY

-   1. Coffin et al., “The discovery of reverse transcriptase,” Ann.     Rev. Virol. 3(1): 29-51 (2016). -   2. Hogrefe et al., “Mutant reverse transcriptase and methods of     use,” U.S. Pat. No. 9,783,791. -   3. Kotewicz et al., “Cloned genes encoding reverse transcriptase     lacking RNase H activity,” U.S. Pat. No. 5,405,776. -   4. Kotewicz et al., “Isolation of cloned Moloney murine leukemia     virus reverse transcriptase lacking ribonuclease H activity,”     Nucleic Acids Res. 16(1): 265-77 (1988). -   5. Rogers et al., “Novel Reverse Transcriptases for Use in High     Temperature Nucleic Acid Synthesis.” U.S. Patent Application     Publication No. US 2015/0210989 A1. 

What is claimed is: 1: An isolated Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutant comprising the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) threonine to lysine at position 306 (T306K); (i) a valine to asparagine at position 433 (V433N); (j) a valine to arginine at position 433 (V433R); (k) an isoleucine to glutamic acid at position 593 (1593E); or (l) an isoleucine to tryptophan at position 593 (I593W). 2: The isolated MMLV RTase mutant of claim 1, wherein the amino acid sequence of the MMLV RTase mutant comprises the amino acid substitutions: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine substitution at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine substitution at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) a threonine to lysine substitution at position 306 (T306K); (i) a valine to asparagine substitution at position 433 (V433N); and (j) an isoleucine to tryptophan substitution at position 593 (I593W). 3: The isolated MMLV RTase mutant of claim 1, wherein the amino acid sequence of the MMLV RTase mutant comprises the amino acid substitutions: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine substitution at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine substitution at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) a threonine to lysine substitution at position 306 (T306K); (i) a valine to arginine substitution at position 433 (V433R); and (j) an isoleucine to glutamic acid substitution at position 593 (I593E). 4: The isolated MMLV Rtase mutant of claim 1, wherein the MMLV RTase mutant comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 716-731. 5: The isolated MMLV Rtase mutant of claim 4, wherein the MMLV RTase mutant comprises an amino acid sequence as set forth in SEQ ID NO:
 725. 6: The isolated MMLV Rtase mutant of claim 4, wherein the MMLV RTase mutant comprises an amino acid sequence as set forth in SEQ ID NO:
 731. 7: The MMLV RTase mutant of any one of claims 1 to 6, wherein the MMLV RTase mutant lacks RNase H activity. 8: The MMLV RTase mutant of any one of claims 1 to 6, wherein the MMLV RTase mutant possesses at least one of the following characteristics: enhanced DNA synthesis, increased fidelity, or enhanced thermostability. 9: An isolated nucleic acid molecule comprising a nucleotide sequence encoding the MMLV Rtase mutant of any one of claims 1 to
 6. 10: A composition comprising the isolated MMLV RTase mutant of any one of claims 1 to
 6. 11: The composition of claim 10, wherein the isolated MMLV RTase mutant lacks RNase H activity. 12: The composition of claim 11, wherein the isolated MMLV RTase mutant possesses at least one of the following characteristics: enhanced DNA synthesis, increased fidelity, or enhanced thermostability. 13: A kit comprising the isolated MMLV RTase mutant of mutant of any one of claims 1 to
 6. 14: The kit of claim 13, wherein the isolated MMLV RTase mutant lacks RNAse H activity. 15: The kit of claim 14, wherein the isolated MMLV RTase mutant possesses at least one of the following characteristics: enhanced DNA synthesis, increased fidelity, or enhanced thermostability. 16: A method for synthesizing complementary deoxyribonucleic acid (cDNA) comprising: (a) providing the isolated MMLV RTase mutant of any one of claims 1 to 6; and (b) contacting the isolated MMLV RTase mutant with a nucleic acid template to permit synthesis of cDNA. 17: A method for performing reverse transcription-polymerase chain reaction (RT-PCR) comprising: (a) providing the isolated MMLV RTase mutant of any one of claims 1 to 6; and (b) contacting the isolated MMLV RTase mutant with a nucleic acid template to replicate and amplify the nucleic acid template. 